Enhancement of immune responses by 4-1bb-binding agents

ABSTRACT

This invention features methods of enhancing immune responses in mammalian subjects and in vitro methods of enhancing the response of a T cell. Also embodied by the invention are methods of receiving and preventing the induction of energy in T cells.

This application claims priority under 35 U.S.C. Section 119 ofInternational Application PCT/US02/32364 filed Oct. 9, 2002 and U.S.Provisional application Ser. No. 60/328,004 filed Oct. 9, 2001.

The research described in this application was supported in part bygrant R0.1 CA79915 from the National Cancer Institute of the NationalInstitutes of Health. The government may have certain rights in theinvention.

TECHNICAL FIELD

This invention relates to immunoregulation, and more particularly to Tcell response regulation.

BACKGROUND

Mammalian T lymphocytes recognize antigenic-peptides bound to majorhistocompatibility complex (MHC) molecules on the surface of antigenpresenting cells (APC). The antigenic peptides are generated byproteolytic degradation of protein antigens within the APC. Theinteraction of the T cells with the APC and the subsequent response ofthe T cells are qualitatively and quantitatively regulated byinteractions between cell surface receptors on the T cells with bothsoluble mediators and ligands on the surface of APC.

SUMMARY

The inventors have discovered that treatment of mice bearing a weaklyimmunogenic tumor with an agonistic antibody specific for murine 4-1BB(also known as CD137) and a peptide fragment of a polypeptide expressedby the tumor resulted in regression of the tumor. In addition, treatmentof mice bearing a second weakly immunogenic tumor with the same 4-1BBantibody and autologous dendritic cells “primed” in vitro with cells ofthe tumor resulted in regression of the tumor. Moreover, the inventorshave found that signaling via cell-surface 4-1BB molecules and providingan immunogenic stimulus (a) prevents induction of anergy in CD8+ T cellsand (b) reverses already established anergy in the CD8+ T cells. Thusthe invention features a method of enhancing a mammalian immune responsethat involves administering to a mammalian subject (e.g., a cancerpatient) (a) an immunogenic stimulus such as a tumor associatedpeptide-epitope and (b) an agonistic 4-1BB-binding agent (e.g., a4-1BB-specific antibody). The invention also features an in vitro methodof enhancing the response of a T cell in which a population of cellscontaining a T cell is incubated with (a) an immunogenic stimulus suchas a peptide-epitope from an infectious microoganism and (b) anagonistic 4-1BB-binding agent (e.g., the 4-1BB ligand).

More specifically, the invention features a method of generating anenhanced immune response in a subject. The method involves administeringto the subject: (a) an immunogenic stimulus; and (b) an agonistic4-1BB-binding agent. The subject can be a human, e.g., a human cancerpatient. The immune response enhanced can be a response of a T cell,e.g., a CD8+ T cell or a CD4+ T cell.

The invention also embraces an in vitro method of activating a T cell,e.g., a CD8+ T cell or a CD4+ T cell. This method involves: (a)providing a cell sample comprising a T cell; and (b) culturing the cellsample with an immunogenic stimulus and an agonistic 4-1BB-bindingagent.

Another aspect of the invention is a method of preventing induction ofanergy or of reversing anergy in a T cell; the method includescontacting the T cell with: (a) an immunogenic stimulus; and (b) anagonistic 4-1BB-binding agent. The contacting can be in vitro or the Tcell can be in a mammal (e.g. a human). The contacting can includeadministering to the mammal: (a) the immunogenic stimulus and theagonistic 4-1BB-binding agent; (b) a nucleic acid encoding theimmunogenic stimulus and the agonistic 4-1BB-binding agent; (c) theimmunogenic stimulus and a nucleic acid encoding the agonistic 4-1BBbinding agent; or (d) a nucleic acid encoding the immunogenic stimulusand a nucleic acid encoding the agonistic 4-1BB binding agent. Thecontacting can alternatively include administering to the mammal anucleic acid encoding the immunogenic stimulus and a nucleic acidencoding the 4-1BB binding agent, the nucleic acid encoding theimmunogenic stimulus and the nucleic acid encoding the 4-1BB bindingagent being in the same nucleic acid molecule. Moreover, the method caninclude administering a cell transfected or transduced with a nucleicacid encoding the immunogenic stimulus or the 4-1BB-binding agent to themammal, the cell being a cell, or a progeny of a cell, that prior to thetransfection or the transduction, was obtained from the mammal.

In the methods of the invention, the agonistic 4-1BB binding agent canbe, for example, (1) an antibody that binds to 4-1BB or (2) the naturalligand for 4-1BB (4-1BBL) or a functional fragment thereof. Theimmunogenic stimulus can be a (a) a tumor-associated antigen (TAA) or(b) a functional fragment of a TAA and it can be a polypeptide. The TAAcan be a molecule produced by a leukemia, a lymphoma, a neurologicalcancer, a melanoma, a breast cancer, a lung cancer, a head and neckcancer, a gastrointestinal cancer, a liver cancer, a pancreatic cancer,a genitourinary cancer, a prostate cancer, a renal cell cancer, a bonecancer, or a vascular cancer cell. The immunogenic stimulus can be adendritic cell that has a major histocompatibility complex (MHC)molecule with peptide-epitope bound thereto, the peptide-epitope being afragment of a TAA or a fragment of a polypeptide produced by aninfectious microorganism. The MHC molecule can be a MHC class I moleculeor a MHC class II molecule. The immunogenic stimulus can be also be ahybrid cell, e.g., a fusion product of a tumor cell and a dendriticcell. In addition, the immunogenic stimulus can be a tumor cell, a tumorcell lysate, a TAA, a peptide-epitope of a TAA, or a heat shock proteinbound to peptide-epitope of protein expressed by a tumor cell. Theimmunogenic stimulus can also be a dendritic cell that has beenincubated with tumor cells, a tumor cell lysate, a TAA, apeptide-epitope of a TAA, or a heat shock protein bound topeptide-epitope of protein expressed by a tumor cell. Where theimmunogenic stimulus is or contains a tumor cell, an APC, or a hybridcell, the cell can be transfected with or transformed with a nucleicacid encoding a cytokine or a growth factor, e.g., granulocytemacrophage-colony stimulating factor (GM-CSF).

Alternatively, the immunogenic stimulus can be a molecule produced by aninfectious microorganism, e.g., a virus such as a retrovirus, abacterium, a fungus, or a protozoan parasite.

As used herein, an “enhanced immune response” is obtained byadministering to a subject an immunogenic stimulus and an agonistic4-1BB-binding agent. In the absence of administration of an agonistic4-1BB-binding agent, an appropriate immunogenic stimulus eitherstimulates no immune response in the subject or it stimulates an immuneresponse in the subject that is detectably lower than a responsestimulated by administration of the immunogenic stimulus and anagonistic 4-1BB-binding agent.

As used herein, an “anergic T cell” or an “anergized T cell” is a T cellwhose ability to respond to an immunogenic stimulus, with respect to atleast one activity of the T cell, has been partially or completelyinhibited. Thus, for example, an anergic or anergized T cell may lack orhave a decreased ability to proliferate and produce interleukin-2 inresponse to an immunogenic stimulus but its ability to produceinterferon-γ in response to the immunogenic stimulus may besubstantially intact. Alternatively, an anergic or anergized T cell maydisplay a lack or a decrease in all the functional activities elicitedby an immunogenic stimulus in such a T cell.

As used herein, “reversing” anergy in a T cell means fully or partiallyrestoring the ability of the T cell to perform one or more functions inresponse to an immunogenic stimulus.

As used herein, “preventing the induction of” anergy in a T cell meansfully or partially inhibiting the induction of anergy in the T cell.

As used herein, an “agonistic 4-1BB-binding agent” is a substance thatupon binding to a 4-1BB molecule on a target cell (e.g., a T cell)enhances the response of the target cell to an immunogenic stimulus.

As used herein, a “functional fragment of a tumor-associated antigen(TAA)” is a fragment of a TAA shorter than the full-length TAA but withgreater than 10% (e.g., greater than 10%, greater than 20%, greater than30%, greater than 40%, greater than 50%, greater than 60%, greater than70%, greater than 80%, greater than 90%, greater than 95%, greater than98%, greater than 99%, greater than 99.5%, or 100% or more) of theability of the full-length TAA to activate an immune response in thepresence or absence of an agonistic 4-1BB-binding agent. In TAA that arepolypeptides, a “full-length” TAA is the mature TAA, i.e. thepolypeptide lacking its native signal sequence.

As used herein, a “peptide-epitope” of a polypeptide is a fragment of apolypeptide that binds to a major histocompatibility complex (MHC)molecule and is recognized in the form of a complex with the MHCmolecule by an antigen specific receptor on a T cell (TCR). MHCmolecules can be class I or class II MHC molecules.

“Polypeptide” and “protein” are used interchangeably and mean anypeptide-linked chain of amino acids, regardless of length orpost-translational modification.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention. All publications, patentapplications, patents and other references mentioned herein areincorporated by reference in their entirety. The materials, methods, andexamples disclosed herein are illustrative only and not intended to belimiting.

Other features and advantages of the invention, e.g., enhancing immuneresponses in mammalian subjects, will be apparent from the followingdescription, from the drawings and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a flow cytometry (FFC) histogram showing the binding of mAb2A (unfilled profile with solid line (labeled “2A”) and unfilled profilewith dashed line (labeled “2A+4-1BB”)) or an isotype control antibody(filled profile) to nylon wool-purified murine T cells stimulated for 24hours with immobilized antibodies specific for CD3 and CD28. Thestaining reactions were performed in the absence (unfilled profile withsolid line and filled profile) or presence (unfilled profile with dashedline) of the murine 4-1BBIg fusion protein.

FIG. 1B is a FFC histogram showing binding of mAb 2A (unfilled profile(labeled “2A”)), a commercially available mAb specific for murine 4-1BB(unfilled profile labeled “1AH2”), or an isotype control antibody(filled profile) to S49.1 murine T lymphoma cells.

FIG. 1C is a line graph showing the proliferation (“³H-TdRIncorporation”) in counts per minute (“cpm×10⁵”) of nylon wool-purifiedmurine T cells stimulated with antibody specific for mouse CD3 (coatedonto well-bottoms of 96-well tissue culture plates at a concentration of0.1 μg/ml) and either mAb 2A (open circles) or rat IgG (filled circles),each coated onto the well-bottoms of the 96-well tissue culture platesat the indicated concentrations.

FIG. 2 is a series of line graphs showing the growth (“Average TumorDiameter”) of tumors in mice injected subcutaneously (s.c.) with C3tumor cells (bottom panels) or EL4E7 tumor cells (top panels) on day 0.Seven days after injection of the tumor cells the mice were injectedwith either mAb 2A (right panels) or rat IgG (left panels). Each linerepresents a single mouse.

FIGS. 3A and 3B are line graphs showing the cytolytic activity (“%Lysis”) against the indicated target cells of effector cells at variouseffector target cell ratios (“E:T Ratio”). Mice were injected s.c. witheither 1×10⁶ C3 (FIG. 3A) or 4×10⁶ EL4E7 (FIG. 3B) tumor cells on day 0.The mice were injected with 100 μg of either mAb 2A (open circles) orrat IgG (closed circles) on days 1 and 4. On day 7 the mice weresacrificed, tumor-draining lymph nodes (TDLN) were harvested, and thecells obtained from the TDLN were stimulated in vitro for four days withirradiated C3 cells (FIG. 3A) or irradiated EL4E7 cells (FIG. 3B). TDLNfrom 2-3 mice in each group were pooled. Cells harvested from thestimulating cultures were tested for cytolytic activity in a standard 4hour ⁵¹Cr-release assay against EL4, EL4E7, RMA-S, E7 peptide-pulsedRMA-S (“RMA-S+E7”), or C3 target cells.

FIG. 4A is a series of two-dimensional FFC histograms showing thebinding to cells (obtained from cultures set up as described for FIG. 3)of mAb specific for CD8 and tetramers of H-2D^(b) class molecules boundto the E7 (49-57) peptide (“D^(b)/E7”). Data obtained with cells frommice injected with C3 cells are shown in the top two panels and dataobtained with cells from mice injected with EL4E7 cells are shown in thebottom two panels. The mice were injected with either the 2A mAb (leftpanels) or rat IgG (right panels). The circles within the histogramsindicate the area gated to detect cells binding both the CD8-specificmAb and the D^(b)/E7 tetramer.

FIG. 4B is a bar graph showing the relative number of cells (in the cellpopulations described in FIG. 4A) binding both the CD8-specific mAb andthe D^(b)/E7 tetramer (“% CD8⁺/E7 Tetramer⁺”). Thus, the cells were fromcultures set up from mice injected with: C3 cells and rat IgG (“C3+RatIgG”: top left histogram in FIG. 4A); C3 cells and mAb 2A (“C3+2A”; topright histogram in FIG. 4A); EL4E7 cells and rat IgG (“EL4E7+Rat IgG”;bottom left histogram in FIG. 4A); and EL4E7 cells and mAb 2A(“EL4E7+2A”; bottom right histogram in FIG. 4A).

FIGS. 5A and 5B are bar graphs showing the relative number of cellsbinding both the CD8-specific mAb and the D^(b)/E7 tetramer (“% CD8⁺/E7Tetramer⁺”) in lymph nodes from mice treated as follows. Normal mice(FIG. 5B) or mice injected s.c. (in one flank) seven days earlier with1×10³ C3 cells (FIG. 5A) were immunized by s.c. injection in thecontralateral flank with 50 μg of the E7 (49-57) peptide (or the controlVp2 peptide) emulsified in incomplete Freund's adjuvant (IFA). The miceinjected with the Vp2 peptide and some of the mice injected with the E7(49-57) peptide were injected i.p. with 100 μg of mAb 2A (“Vp2+2A” and“E7+2A”. respectively) on the day following, and four days after,immunization. Other mice injected with the E7 (49-57) peptide wereinjected i.p. with 100 μg of rat IgG (“E7 +Rat IgG”) on the dayfollowing, and four days after, immunization. Seven days afterimmunization with peptide the mice were sacrificed and lymph nodesdraining the site of immunization were removed. Lymph nodes from 2-3mice per group were pooled. Cell suspensions were prepared from thelymph nodes and the relative number of cells binding both CD8 and theD^(b)/E7 tetramer (“% CD8⁺/E7 Tetramer⁺”) were determined as describedabove for FIG. 4.

FIG. 6 is a scattergram showing (in mice treated as described below) (i)the number of mice that never developed palpable tumors and (ii) thenumber of weeks after injection of tumor cells that had elapsed when theindicated mice were sacrificed, i.e., when the tumors in the miceattained an average diameter of 15 mm. The mice were treated as follows.All mice were injected s.c. with 1×10⁶ C3 cells on day 0. On day 7, halfthe mice were immunized s.c. with the E7 (49-57) peptide and the otherhalf with the control Vp2 (121-130) peptide emulsified in IFA. On days 7and 10, the mice were injected i.p. with either 100 μg of mAb 2A (“2A”)or rat IgG (“Rat IgG”). Tumor size was assessed weekly for each mouseand the mice were sacrificed when the average tumor diameter reached 15mm (open circles). Mice bearing tumors less than 15 mm in average tumordiameter at the termination of the experiment (week 13) are indicated byopen circles. Mice that never developed palpable tumors are indicated byclosed circles in the top part of the figure.

FIGS. 7A and 7B are survival curves showing the survival (“% Survival”)of mice treated as follows. Mice were injected intravenously (i.v.) witheither 1×10⁶ TC-1 cells (FIG. 7A) or 1×10⁵ B16-F10 cells on day 0. Onday 3, the mice were injected s.c. with either the control OVA peptideor (circles), the E7 (49-57) (“E7”) peptide (triangles in FIG. 7A), orthe trp-2 peptide (triangles in FIG. 7B) (50 μg of the indicate peptideemulsified in IFA in each flank). On day 3 and on day 6 the mice wereinjected with 100 μg of either the 2A mAb (“2A”) or rat IgG. The micewere observed daily for the duration of the experiment. Survival datafrom two identically performed experiments were combined.

FIG. 8 is a series of line graphs showing the growth of tumors in miceinjected s.c. (in one flank) with SCCVII tumor cells on day 0. On day 4,the mice were divided into four groups (5 mice per group) that weretreated as follows. On days 4 and 11, the mice in the first two groupswere injected s.c. in the contralateral flank with dendritic cells“primed” in vitro with SCCVII cells (bottom panels). On days 4, 7, 11and 14, the mice in the first group were injected i.p. with 100 μg ofrat IgG (bottom right panel; “RAT Ig DC/XRT”) and the mice in the secondgroup were injected i.p. with 100 μg of mAb 2A (bottom left panel; “41BB/2A DC/XRT”). On days 4, 7, 11, and 14 the mice in third and fourthgroup were injected with 100 μg of rat IgG (top right panel; “RAT Ig”)and 100 μg of mAb 2A (top left panel; “4 1BB/2A”), respectively. Tumorsize (mean diameter; “Mean Tumor Size”) was monitored. Each line in thegraphs represents a single mouse.

FIG. 9 is a line graph showing the proliferation (“³H-Thyincorporation”) in counts per minute (“cpm”) of nylon wool purifiedhuman T cells stimulated with antibody specific for human CD3 (coatedonto well-bottoms of 96-well tissue culture plates at the indicatedconcentrations) and either mAb 5.9 (“α-h41BB (5.9)”), mAb 5.10 (“α-h41BB(5.1 0)”), a commercially available antibody specific for human 4-1BB(“α-h41BB”), or mouse IgG (“mIgG”), each coated onto the well-bottoms of96-well tissue culture plates at a concentration of 10 μg/ml.

FIG. 10 is a survival curve showing the survival (“% Survival”) of B6mice treated as follows. Mice were injected intravenously (i.v.) with5×10⁵ B16-F10 cells on day 0. On days 3, 7, and lithe mice were injecteds.c. with irradiated B16-F10 cells expressing recombinant GM-CSF(triangles) or were not treated (circles). On days 4, 7, 10, 12, and 15,the mice were injected with 100 μg of either the 2A mAb (“2A”) (filledcircles and triangles) or rat IgG (unfilled circles and triangles). Themice were observed daily for the duration of the experiment.

FIG. 11 is a series of one- and two-dimensional FFC histograms showingthe forward light scatter (“FSC”) and the relative proportion ofCD69-expressing CD25-expressing cells in CD8+, OVA tetramer+pooled lymphnode and splenic T cells from C57BL/6 (B6) mice that had been injectedwith OT-1 transgenic mouse lymphoid cells and either a control peptide(top histograms) or the OVA peptide (bottom histograms). Thetwo-dimensional FFC histograms (left panels) show the relative numbersof CD8-expressing cells (x-axis) and OT-1 transgenic T cell receptor(TCR)-expressing (OVA tetramer+) (y-axis). Cells expressing CD8 and thetransgenic OT-1 TCR were gated (as indicated by the circle in the toptwo-dimensional FFC histogram and the ellipse in the bottomtwo-dimensional FFC histogram) and the gated populations were analyzedfor FSC (shown in the first one-dimensional FFC histograms) and therelative proportions of CD69-expressing cells (shown in the secondone-dimensional FFC histograms) and CD25-expressing cells (shown in thethird one-dimensional FFC histograms). The numbers in thetwo-dimensional FFC histograms indicate the proportions ofCD8-expressing, OT-1 transgenic TCR-expressing cells in the pooled lymphnode and spleen cells from the two groups of mice.

FIG. 12A is a pair of bar graphs showing means (and standard deviations)of the total numbers (“Total OT-1”) of OT-1 transgenic TCR-expressingcells in pooled lymph node and spleen cells from individual B6 mice (3per group) that were injected with OT-1 transgenic mouse lymphoid cellsand either phosphate buffered saline (PBS) (left panel; “Naïve”) or theOVA peptide (right panel; “Anergic”), followed ten days later by achallenge with either a control peptide (“−”) or the OVA peptide (“+”).The mice were sacrificed, the spleens and lymph nodes removed, andpooled cell suspensions prepared separately from each mouse two daysafter the challenge. The cells were analyzed for expression of the OT-1transgenic TCR and CD8 by FFC and the total numbers of OT-1 transgenicTCR-expressing cells in the various cell preparations were calculated.

FIG. 12B is a pair of FFC histograms showing the relative proportions ofCD69-(left panel) and CD25-(right panel) expressing cells from the micedescribed for FIG. 12A that had received an initial injection of andbeen challenged with the OVA peptide. Cells stained with CD69 or CD25are shown by the unfilled profiles and those stained with an isotypecontrol antibody are shown by the filled profiles.

FIG. 12C is a bar graph showing the amount of interferon-γ (IFN-γ) (pgper ml per 1,000 OT-1 transgenic TCR-expressing cells) produced (afterculturing for 72 hours in the presence of the OVA peptide) by theanergic (unfilled bar) and naïve (filled bar) cells described for FIG.12A that had initially been injected with PBS (“Naïve”) or the OVApeptide (“Anergic”) and then challenged with the OVA peptide.

FIG. 13A is a line graph showing the means (and standard deviations) ofthe total numbers of OT-1 transgenic TCR-expressing cells (“OT-1cells/mouse”) in pooled lymph node and spleen cells from individual B6mice (3 mice per group) at various times (“Time (days)”) after injectionwith OT-1 transgenic mouse lymphoid cells and either PBS or the OVApeptide (“OVA”). On the day of OVA peptide (or PBS) injection, and againthree days later, the mice were injected with either control rat IgG(“rat IgG”) or anti-CD 137 monoclonal antibody (mAb) (“2A”).

FIG. 13B is a pair of bar graphs showing the relative proliferation invitro (left panel) and amount of interleukin-2 (IL-2) (right panel)produced in vitro by spleen cells prepared from the individual B6 mice(3 mice per group) described for FIG. 13A 10 days following injectionwith the OVA peptide (or PBS). The numbers of OT-1 transgenicTCR-expressing cells in the spleen cell preparations were calculated andthe data for proliferation are expressed as means (and standarddeviations) of the counts per minute (cpm) of [³H]-thymidineincorporated per 1,000 OT-1 transgenic TCR-expressing cells in the assaycultures (“Δcpm/OT-1 (10³)”) and the data for IL-2 production areexpressed as the means (and standard deviations) of the amounts (inpg/ml) of IL-2 produced per OT-1 transgenic TCR-expressing cell (“IL-2(pg ml⁻¹/10³ OT-1”) in the assay cultures.

FIG. 13C is a pair of FFC histograms showing the relative proportion ofcells staining with an anti-VLA mAb (unfilled profiles) or isotypecontrol mAb (filled profiles) in spleen cells from the mice describedfor FIG. 13A that had been injected with the OVA peptide after achallenge (ten days after the initial injection with the OVA peptide)with the OVA peptide (right panel) or a control peptide.

FIG. 14A is a line graph showing the means (and standard deviations) ofthe total numbers of OT-1 transgenic TCR-expressing cells (“OT-1cells/mouse”) in pooled lymph node and spleen cells from individual B6mice (3 mice per group) that had been injected with OT-1 transgenicmouse lymphoid cells and the OVA peptide and ten days after the OVApeptide injection challenged with either the OVA peptide (“OVA”) or acontrol peptide (“control peptide”) and injected with either control ratIgG (“rat IgG”) or anti-CD137 monoclonal antibody (mAb) (“2A”). Assayswere performed at the indicated days after the challenge (“Time (day)”).

FIG. 14B is a bar graph showing the means (and standard deviations) ofthe total numbers of OT-1 transgenic TCR-expressing cells (“OT-1cells/mouse”) in pooled lymph node and spleen cells from individual B6mice (3 mice per group) that had been injected with OT-1 transgenicmouse lymphoid cells and the OVA peptide and ten days after the OVApeptide injection challenged with the indicated combinations of the OVApeptide (“OVA”) or a control peptide (“control peptide”) and control ratIgG (“rat IgG”) or anti-CD137 mAb (“2A”). The assay was performed 3 daysafter the challenges.

FIG. 14C is a pair of histograms showing the relative proportion ofcells staining with an anti-CD25 mAb (unfilled profiles) or an isotypecontrol mAb (filled profiles) in the cells from the mice described forFIG. 14B that were challenged with OVA and injected with eitheranti-CD137 mAb (“2A”; right panel) or control rat IgG (“rat IgG”; leftpanel).

FIG. 14D is a pair of line graphs showing the cytotoxic activity (“%Lysis”) of various cell populations against EL4 target cells pulsed withthe OVA peptide (left panel) and control EL4 target cells (right panel).B6 mice were injected with OT-1 transgenic mouse lymphoid cells and theOVA peptide and ten days after the OVA peptide injection were challengedwith the OVA peptide (“OVA”) and injected with anti-CD137 mAb; five daysafter the challenge and mAb injection, cells expressing the OT-1transgenic TCR were sorted by FACS and used as effector cells (“Anergic(OVA+2A)”) in the cytotoxicity assays. Two control OT-1 transgenicTCR-expressing cell populations were also FACS sorted and tested. Onepopulation was from mice that had been treated identically to the firstdescribed population except that, instead of receiving an initialinjection of the OVA peptide, the mice received an injection of acontrol peptide (“OVA+2A”). A second population was obtained from micethat had been treated identically to the first described populationexcept that, instead of receiving an initial injection of the OVApeptide, the mice received an injection of a control peptide, and,instead of being injected with anti-CD 137 mAb at the time of challengewith the OVA peptide, the mice were injected with control rat IgG(“OVA+rat IgG”). An additional control effector population consisted ofOT-1 transgenic TCR-expressing cells sorted by FACS from spleen andlymph nodes of untreated OT-1 TCR transgenic mice (“Naïve”).

FIG. 15A is a series of line graphs showing the incidence of tumors inDBA/2 mice after immunization with either incomplete Freund's adjuvantalone (“IFA”) or with IFA and a P1A peptide epitope (“P1A”) andchallenge with P815R tumor cells. On the day of immunization, and againthree days later, P1A peptide-immunized mice were injected with eitheranti-CD137 mAb (“2A”) or control rat IgG (“rat IgG”). All the mice werechallenged with the P815R tumor cells 10 days after immunization andwere observed for tumor incidence and regression at the indicated timepoints after tumor challenge (“Days after tumor inoculation”).

FIG. 15B is a pair of line graphs showing the incidence of tumors inDBA/2 mice after immunization with IFA and a P1A peptide epitope andchallenge with P815R tumor cells. The mice were immunized with IFA andthe P1A peptide and were challenged with the P815R tumor cells 10 daysafter immunization. Three days after tumor challenge, and again threedays later, the mice were injected with either anti-CD 137 mAb (“2A”) orcontrol rat IgG (“rat IgG”) and were observed for tumor incidence andregression at the indicated time points after tumor challenge (“Daysafter tumor inoculation”).

DETAILED DESCRIPTION

The invention is based on the finding that treatment of mice bearing aweakly immunogenic tumor with an agonistic antibody specific for murine4-1BB and a peptide fragment of a polypeptide expressed by the tumorresulted in regression of the tumor. In addition, treatment of micebearing a second weakly immunogenic tumor with the same 4-1BB antibodyand autologous dendritic cells “primed” in vitro with cells of the tumorresulted in regression of the tumor. The inventors have also discoveredthat providing an immunogenic stimulus and a 4-1BB-mediated signal to aCD8+ T cell not only prevents induction of anergy in a CD8+ T cell, butcan also reverse, partially or completely, already established anergy ina CD8+ T cell. These findings indicate that treatment of mammaliansubjects having (or being at risk of having) cancer or an infectiousdisease with an. agonistic agent that binds to the 4-1BB molecule (e.g.,the 4-1BB ligand or an agonistic antibody specific for 4-1BB) and animmunogenic stimulus specific for the relevant cancer or infectiousmicroorganism will result in an enhanced immune response to the canceror infectious agent. Such an enhanced immune response will preferably,though not necessarily, be prophylactic and/or therapeutic for therelevant disease.

Methods of Activating an Immune Response

The invention features methods of activating mammalian immune responsesin which cells of the immune system are exposed to (a) an immunogenicstimulus and (b) an agonistic 4-1BB-binding agent. Exposure of the cellsto the immunogenic stimulus can occur before, during, or after exposureto the 4-1BB binding agent. The two exposures will preferably besubstantially simultaneous.

Responses that are enhanced by the methods of the invention can be anyimmune response. The responses enhanced are preferably T cell responses.However, snce antibody-producing responses of B cells are generallydependent on helper activity of activated CD4+ T cells, enhancement of aCD4+ T cell helper cell response can indirectly result in enhancement ofa B cell antibody response. Similarly, the activities of other cells ofthe immune system (e.g., monocytes/macrophages, granulocytes (e.g.,neutrophils), and natural killer cells) are regulated by T cells. Thusthe methods of the invention can be used to enhance responses of any orall of these cell types.

The invention also provides methods for preventing induction of anergyin T cells or reversing anergy in T cells already rendered anergic. Inthese methods the relevant T cells are contacted with an immmunogenicstimulus and a 4-1BB-binding agent. Contacting of the T cells with theimmunogenic stimulus can occur before, during, or after contacting the Tcells with the 4-1BB binding agent.

As used herein, an “immunogenic stimulus” is a stimulus delivered to a Tcell via the antigen-specific T cell receptor (TCR) expressed on thesurface of the T cell. More commonly, but not necessarily, such astimulus is provided in the form of an antigen for which the TCR isspecific. While such antigens will generally be protein, they can alsobe carbohydrates, lipids, nucleic acids or hybrid molecules havingcomponents of two or more of these molecule types, e.g., glycoproteinsor lipoproteins. However, the immunogenic stimulus can also be providedby other agonistic TCR ligands such as antibodies specific for TCRcomponents (e.g., TCR α-chain or β-chain variable regions) or antibodiesspecific for the TCR-associated CD3 complex. Immunogenic stimuli (asused herein) do not include antigen-non-specific stimuli provided by,for example, cytokines (e.g., interleukin-12), growth factors,co-stimulatory molecules, or adhesion molecules. While such stimuli canbe exploited in the methods of the invention, they do not constitute therequired immunogenic stimulus. Antigens useful as immunogenic stimuliinclude alloantigens (e.g., a MHC alloantigen) on, for example, anantigen presenting cell (APC) (e.g., a dendritic cell (DC), amacrophage, a monocyte, or a B cell). DC of interest are interdigitatingDC and not follicular DC; follicular DC present antigen to B cells. Forconvenience, interdigitating DC are referred to herein as DC. Methods ofisolating DC from tissues such as blood, bone marrow, spleen, or lymphnode are known in the art, as are methods of generating them in vitrofrom precursor cells in such tissues. Also useful as immunogenic stimuliare polypeptide antigens and peptide-epitopes derived from them.Unprocessed polypeptides are processed by APC into peptide-epitopes thatare presented to responsive T cells in the form of molecular complexeswith MHC molecules on the surface of the APC. Useful immunogenic stimulialso include a source of antigen such as a lysate of either tumor cellsor cells infected with an infectious microorganism of interest. APC(e.g., DC) pre-exposed (e.g., by coculturing) to antigenic polypeptides,peptide-epitopes of such polypeptides or lysates of tumor (or infectedcells) can also be used as immunogenic stimuli. Such APC can also be“primed” with antigen by culture with a cancer cell or infected cell ofinterest; the cancer or infected cells can optionally be irradiated orheated (e.g., boiled) prior to the priming culture. In addition, APC(especially DC) can be “primed” with either total RNA, mRNA, or isolatedTAA-encoding RNA.

Alternatively, antigen as an immunogenic stimulus be provided in theform of cells (e.g., tumor cells or infected cells producing the antigenof interest). In addition, immunogenic stimuli can be provided in theform of cell hybrids formed by fusing APC (e.g., DC) with tumor cells[Gong et al. (2000) Proc. Natl. Acad. Sci. USA 97(6):2716-2718; Gong etal. (1997) Nature Medicine 3(5):558-561; Gong et al. (2000) J. Immunol.165(3):1705-1711] or infected cells of interest. Methods of fusing cells(e.g., by polyethylene glycol, viral fusogenic membrane glycoproteins,or electrofusion) are known in the art. In discussing these cellhybrids, the tumor or infected cell partners will be referred to as theimmunogenic cells (IC). Cells or cell hybrids can be used (asimmunogenic stimuli) untreated or they can be metabolically inhibited(e.g., by irradiation or exposure to a drug such as mitomycin-C) so asto substantially ablate their ability to divide. Tumor or infected cellsused per se as an immunogenic stimulus or as IC for the production ofcell hybrids will preferably, but not necessarily, be derived from thesame donor as that of the T cell. Where the cells are from a differentdonor, they will preferably share one MHC haplotype with the T cell. APCused to form cell hybrids will also preferably, but not necessarily, bederived from the same donor as the T cell. In the production of cellhybrids, either the APC or the IC will be preferably be from, orMHC-compatible with, the donor of the T cell. Alternatively, the APCand/or the IC can share one MHC haplotype (i.e., be semi-allogeneic)with the donor of the T cell. However, as the cells or hybrids used asimmunogenic stimuli will frequently be used in the presence of APC ofthe T cell donor (e.g., in in vivo applications), they can be fully MHCincompatible with the T cell.

Also useful as immunogenic stimuli are heat shock proteins bound toantigenic peptide-epitopes derived from antigens (e.g., tumor-associatedantigens or antigens produced by infectious microorganisms) [Srivastava(2000) Nature Immunology 1(5):363-366]. Such complexes of heat shockprotein and antigenic peptide are useful for facilitating or enhancinguptake of antigenic peptides by APC. Heat shock proteins of interestinclude, without limitation, glycoprotein 96 (gp96), heat shock protein(hsp) 90, hsp70, hsp 110, glucose-regulated protein 170 (grp 170) andcalreticulin. Immunogenic stimuli can include one or more (e.g., one,two, three, four, five, six, seven, eight, nine, ten, more) heat shockproteins isolated from tumor cells or infected cells. Such tumor orinfected cells are preferably, but not necessarily, from the samesubject (i) whose immune response is to be enhanced by a method of theinvention or (ii) from whom T cells (whose response is to be enhanced bya method of the invention) were obtained. The tumor or infected cellscan also be obtained, for example, from another individual having thesame or a related tumor-type or infection as the subject. Alternatively,the heat shock protein can be isolated from mammalian cells expressing atranscriptosome prepared from tumor cells or infected cells of interest.

Immunogenic stimuli can be derived from a wide range of infectiousmicroorganisms (e.g., bacteria, fungi including yeasts, viruses, andparasites such as protozoan parasites). Examples of relevantmicroorganisms include, without limitation, Mycobacteria tuberculosis,Salmonella enteriditis, Listeria monocytogenes, M. leprae,Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae,Borrelia burgdorferi, Actinobacillus pleuropneumoniae, Helicobacterpylori, Neisseria meningitidis, Yersinia enterocolitica, Bordetellapertussis, Porphyromonas gingivalis, mycoplasma, Histoplasma capsulatum,Cryptococcus neoformans, Chlamydia trachomatis, Candida albicans,Plasmodium falciparum. Entamoeba histolytica, Toxoplasma brucei,Toxoplasma gondii, Leishmania major, human immunodeficiency virus 1 and2, influenza virus, measles virus, rabies virus, hepatitis virus A, B,and C, rotaviruses, papilloma virus, respiratory syncytial virus, felineimmunodeficiency virus, feline leukemia virus, and simianimmunodeficiency virus. Examples of relevant microbial proteins include,without limitation, the B subunit of heat labile enterotoxin of E. coli[Konieczny et al. (2000) FEMS immunol. Med. Microbiol. 27(4):321-332],heat-shock proteins, e.g., the Y. enterocolitica heat shock protein 60[Konieczny et al. (2000) supra; Mertz et al. (2000) J. Immunol.164(3):1529-1537] and M. tuberculosis heat-shock proteins hsp60 andhsp70, the Chlamydia trachomatis outer membrane protein [Ortiz et at.(2000) Infect. Immun. 68(3):1719-1723], the B. burgdorferi outer surfaceprotein [Chen et at. (1999) Arthritis Rheum. 42(9):1813-1823], the L.major GP63 [White et al. (1999) Vaccine 17(17):2150-2161 (and publishederratum in Vaccine 17(20-21):2755)], the N. meningitidis meningococcalserotype 15 PorB protein [Delvig et al. (1997) Clin. Immunol.Immunopathol. 85(2);134-142], the P. gingivalis 381 fimbrial protein[Ogawa, (1994) J. Med. Microbiol. 41(5):349-358], the E. coli outermembrane protein F [Williams et al. (2000) Infect. Immun.68(5):2535-2545], influenza virus hemagglutinins and neuramindases,retroviral (e.g., HIV) surface glycoproteins (e.g., HIV gp160/120), orretroviral tat or gag proteins. CTL are by virtue of their ability tokill target cells infected with any of a wide variety of intracellularpathogens (e.g., viruses, or intracellular bacteria and protozoans)potent mediators of immunity to such pathogens. Thus, since the methodsof the invention are efficient at enhancing CTL responses, they can beused for prophylaxis and/or or therapy in infections with suchintracellular pathogens. In addition, helper T cells release a widevariety of cytokines that mediate pathogen-destructive inflammatoryreponses.

As indicated above, immunogenic stimuli useful in the invention can beany of a wide variety of tumor cells, APC “primed” with tumor cells,hybrid cells (see above), tumor-associated antigens (TAA),peptide-epitopes of such TAA, and APC “primed” with TAA orpeptide-epitopes of them. As used herein, a “TAA” is a molecule (e.g., aprotein molecule) that is expressed by a tumor cell and either (a)differs qualitatively from its counterpart expressed in normal cells, or(b) is expressed at a higher level in tumor cells than in normal cells.Thus, a TAA can differ (e.g., by one or more amino acid residues wherethe molecule is a protein) from, or it can be identical to, itscounterpart expressed in normal cells. It is preferably not expressed bynormal cells. Alternatively, it is expressed at a level at leasttwo-fold higher (e.g., a two-fold, three-fold, five-fold, ten-fold,20-fold, 40-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, or15,000-fold higher) in a tumor cell than in the tumor cell's normalcounterpart. Examples of relevant tumors that can be used per se or as asource of antigen (see above) include, without limitation, hematologicalcancers such as leukemias and lymphomas, neurological tumors such asastrocytomas or glioblastomas, melanoma, breast cancer, lung cancer,head and neck cancer, gastrointestinal tumors such as gastric or coloncancer, liver cancer, renal cell cancer, pancreatic cancer,genitourinary tumors such ovarian cancer, vaginal cancer, bladdercancer, testicular cancer, prostate cancer or penile cancer, bonetumors, and vascular tumors. Relevant TAA include, without limitation,carcinoembryonic antigen (CEA), prostate specific antigen (PSA), MAGE(melanoma antigen) 1-4, 6 and 12, MUC (mucin) (e.g., MUC-1, MUC-2,etc.), tyrosinase, MART (melanoma antigen), Pmel 17(gp100), GnT-V intronV sequence (N-acetylglucoaminyltransferase V intron V sequence),Prostate Ca psm, PRAME (melanoma antigen), β-catenin, MUM-1-B (melanomaubiquitous mutated gene product), GAGE (melanoma antigen) 1, BAGE(melanoma antigen) 2-10, c-ERB2 (Her2/neu), EBNA (Epstein-Barr Virusnuclear antigen) 1-6, gp75, human papilloma virus (HPV) E6 and B7, p53,lung resistance protein (LRP) Bc1-2, and Ki-67. Both CTL and helper Tcells have been shown to be efficient effectors of tumor immunity.

Where subjects are administered pathogenic agents (e.g., tumor cells,infectious microorganisms, or cells infected with infectious agents) inorder to test for the efficacy of treatment with an agonistic4-1BB-binding agent (optionally given with one or more non-specificagents such as cytokines (see above)), the pathogenic agents do not perse constitute an immunogenic stimulus for the purposes of the invention.Moreover, in a procedure that involves administration of an agonistic4-1BB-binding agent (and optionally one or more non-specific agents suchas cytokines (see above)) to a subject harboring pathogenic agents(e.g., tumor cells, infectious microorganisms, or cells infected withinfectious agents) acquired by, for example, transformation of one ormore cells in the subject or by natural infection, the harboredpathogenic agents do not per se constitute an immunogenic stimulus forthe purposes of the invention.

The agonistic 4-1BB-binding agent can be an antibody specific for 4-1BB.As used herein, the term “antibody” refers not only to whole antibodymolecules, but also to antigen-binding fragments, e.g., Fab, F(ab′)₂,Fv, and single chain Fv fragments. Also included are chimericantibodies. The antibody can be a polyclonal antibody or a monoclonalantibody (mAb)., e.g., the 2A mAb or the 5.9 and 5.10 mAbs describedbelow. Alternatively, the agonistic 4-1BB-binding agent can be thenatural 4-1BB ligand (4-1BBL) or a functional fragment of 4-1BB. As usedherein, a functional fragment of 4-1BB means a fragment of 4-1BB that isshorter than full-length, mature 4-1BBL and has at least 10% (e.g., atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100% or more) of the ability of full-lengthmature 4-1BBL to enhance the response of a T cell to antigen ofinterest. Methods of testing and comparing the ability of molecules toenhance the response of T cells are known to investigators in the field,e.g., methods (or simple modifications of those) described in theExamples. While it is believed that 4-1BB-binding agents (e.g.,antibodies) that bind to a domain of 4-1BB that is identical oroverlapping with a domain to which 4-1BBL binds are not agonistic, theinvention is not limited by any particular mechanism of action. Methodsto test for agonistic activity in a candidate 4-1BB-binding agent wouldbe essentially the same as those referred to above for testing theability of a fragment of 4-1BBL for its ability to enhance the responseof T cell to an immunogenic stimulus and thus well-known to those in theart.

The agonistic 4-1BB-binding agents can be added to the solution (e.g.,blood or culture medium) containing the T cell. Alternatively, it can besecreted by or expressed on the surface of a cell in the vicinity of theT cell, e.g., an APC presenting an alloantigen or a peptide-epitopebound to an MHC molecule on the surface of the APC. Such cells can alsobe tumor cells, infected cells, or the cell hybrids described above.Where the agonistic 4-1BB-binding agent is secreted by or bound to thesurface of a cell, the cell can be, but is not necessarily, the samecell presenting an alloantigen or a peptide-epitope bound to an MHCmolecule to the T cell. The methods of the invention require theprovision of an exogenous source of the 4-1BB-binding agent. It isunderstood that where the agonistic 4-EBB-binding agent used in themethods of the invention is one secreted by or expressed on the surfaceof a cell such as an APC, tumor cell, infected cell, or hybrid cell, itwill not be an agonistic 4-1BB-binding agent (e.g., 4-1BBL) naturallyexpressed by such cells. Where the only source of an agonistic4-1BB-binding agent is that on the surface of or secreted by an APC, theagonistic 4-1BB-binding agent will be encoded by a recombinant 4-1BBencoding nucleic acid molecule in the APC. Moreover, fortuitousadministration to a subject of an agonistic 4-1BB-binding agent presentin, for example, blood, plasma, or serum administered to the subjectfor, e.g., therapeutic purposes, does not per se constituteadministration of an agonistic 4-1BB-binding agent for the purposes ofthe invention. In addition, the fortuitous presence of an agonistic4-1BB-binding agent in culture medium used for immune cell (e.g., Tcell) activating cultures does not per se constitute the agonistic4-1BB-binding agent required to be present in the in vitro methods ofactivating T cells of the invention.

If the activation is in vitro, the 4-1BB binding agent can be bound tothe floor of a relevant culture vessel, e.g. a well of a plasticmicrotiter plate.

The agonistic 4-1BB-binding agent will preferably, but not necessarily,bind to 4-1BB on the surface of a T cell whose response is enhanced bythe methods of the invention. However, 4-1BB is expressed on cells otherthan T cells, e.g., natural killer (NK) cells and monocytes [Melero etal. (1998) Cell. Immunol. 190:167-172; Kienzle et al. (2000) Int.Immunol. 12:73-82]. Thus, by binding to such non-T cells and therebycausing them to, for example, secrete helper cytokines or express ontheir surface costimulatory molecules and/or adhesion molecules, theresponse of the T cell or the response of a bystander cell (e.g., a Bcell cell antibody response) that is “helped” by the T cell can beenhanced. Similarly, binding of a 4-1BB-binding agent to a CD4+ T cellcan overcome anergy in a bystander CD4+ T cell or CD8+ T cell that isalso exposed to an immunogenic stimulus by, for example, the action ofcytokines produced by the CD4+ T cell to which the 4-1BB binding agentbinds. In an analogous manner, binding of a 4-1BB-binding agent to aCD8+ T cell could overcome anergy in a bystander CD8+ T cell or CD4+ Tcell exposed to an immunogenic stimulus.

Short amino acid sequences can act as signals to direct proteins (e.g.,immunogenic stimuli or agonistic 4-1BB-binding agents) to specificintracellular compartments. For example, hydrophobic signal peptides(e.g., MAISGVPVLGFFIIAVLMSAQESWA (SEQ ID NO:1)) are found at the aminoterminus of proteins destined for the ER. While the sequence KFERQ (SEQID NO:2) (and other closely related sequences) is known to targetintracellular polypeptides to lysosomes, other sequences (e.g.,MDDQRDLISNNEQLP (SEQ ID NO:3) direct polypeptides to endosomes. Inaddition, the peptide sequence KDEL (SEQ ID NO:4) has been shown to actas a retention signal for the ER. Each of these signal peptides, or acombination thereof, can be used to traffic, for example the immunogenicstimuli or agonistic 4-1BB-binding agents to appropriate cellularcompartments. Other signal sequences of interest include the HIV_(tat)transduction domain (RKKRRQRR; SEQ ID NO:5), the Antennapediahomeodomain (RQIKIWFPNRRMKWKK; SEQ ID NO:6) and signal sequences derivedfrom fibroblast growth factor [Lin et al. (1995) J. Biol. Chem.220:14255-14258], transportan [Pooga et al. (1998) FASEB J. 12:67-77],and HSV-1 structural protein VP22 [Elliott et al. (1997) Cell88:223-233]. Poly-arginine sequences (of 7 to 15 arginine residues) canalso be used as membrane translocating domains. DNAs encoding thepolypeptides containing targeting signals can be generated by PCR orother standard genetic engineering or synthetic techniques.

The immunogenic stimuli and agonistic 4-1BB-binding agents can have theamino acid sequences of naturally occurring molecules or they can havesubstitutions. Such substitutions will preferably be conservativesubstitutions. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine,glutamine, serine, and threonine; lysine, histidine, and arginine; andphenylalanine and tyrosine.

Polypeptides useful for the invention also include those describedabove, but modified for in vivo use by the addition, at the amino-and/or carboxyl-terminal ends, of a blocking agent to facilitatesurvival of the relevant polypeptide in vivo. This can be useful inthose situations in which the peptide termini tend to be degraded byproteases prior to cellular uptake. Such blocking agents can include,without limitation, additional related or unrelated peptide sequencesthat can be attached to the amino and/or carboxyl terminal residues ofthe peptide to be administered. This can be done either chemicallyduring the synthesis of the peptide or by recombinant DNA technology bymethods familiar to artisans of average skill.

Alternatively, blocking agents such as pyroglutamic acid or othermolecules known in the art can be attached to the amino and/or carboxylterminal residues, or the amino group at the amino terminus or carboxylgroup at the carboxyl terminus can be replaced with a different moiety.Likewise, the peptide compounds can be covalently or noncovalentlycoupled to pharmaceutically acceptable “carrier” proteins prior toadministration.

Also of interest are peptidomimetic compounds that are designed basedupon the amino acid sequences of polypeptides of interest.Peptidomimetic compounds are synthetic compounds having athree-dimensional conformation (i.e., a “peptide motif”) that issubstantially the same as the three-dimensional conformation of aselected peptide. The peptide motif provides the peptidomimetic compoundwith the ability to activate an immune response (in the case ofimmunogenic stimuli) and enhance an immune response (in the case of theagonistic 4-1BB-binding agents). Peptidomimetic compounds can haveadditional characteristics that enhance their in vivo utility, such asincreased cell permeability and prolonged biological half-life.

The peptidomimetics typically have a backbone that is partially orcompletely non-peptide, but with side groups that are identical to theside groups of the amino acid residues that occur in the peptide onwhich the peptidomimetic is based. Several types of chemical bonds,e.g., ester, thioester, thioamide, retroamide, reduced carbonyl,dimethylene and ketomethylene bonds, are known in the art to begenerally useful substitutes for peptide bonds in the construction ofprotease-resistant peptidomimetics.

Molecules useful as immunogenic stimuli and agonistic 4-1BB-bindingagents can be produced by any of a wide range of methods known in theart. They can be purified from natural sources (e.g., from any of thepathogenic agents listed herein). Smaller peptides (fewer than 100 aminoacids long) and other non-protein molecules can be convenientlysynthesized by standard chemical means known to those in the art. Inaddition, both polypeptides and peptides can be manufactured by standardin vitro recombinant DNA techniques and in vivo transgenesis usingnucleotide sequences encoding the appropriate polypeptides or peptides(see Nucleic Acids section below). Methods well-known to those skilledin the art can be used to construct expression vectors containingrelevant coding sequences and appropriate transcriptional/translationalregulatory elements. See, for example, the techniques described inSambrook et al., Molecular Cloning: A Laboratory Manual (2nd Ed.) [ColdSpring Harbor Laboratory, N.Y., 1989], and Ausubel et al., CurrentProtocols in Molecular Biology [Green Publishing Associates and WileyInterscience, N.Y., 1989].

The transcriptional/translational regulatory elements referred to aboveinclude but are not limited to inducible and non-inducible promoters,enhancers, operators and other elements that are known to those skilledin the art and that drive or otherwise regulate gene expression. Suchregulatory elements include but are not limited to the cytomegalovirushCMV immediate early gene, the early or late promoters of SV40adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage A, the controlregions of fd coat protein, the promoter for 3-phosphoglycerate kinase,the promoters of acid phosphatase, and the promoters of the yeastα-mating factors.

The expression systems that may be used for purposes of the inventioninclude but are not limited to microorganisms such as bacteria (forexample, E. coli and B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA, or cosmid DNA expression vectorscontaining a nucleic acid molecules encoding immunogenic stimuli oragonistic 4-1BB-binding agents; yeast (for example, Saccharomyces andPichia) transformed with recombinant yeast expression vectors containinga nucleic acid encoding immunogenic stimuli or agonistic 4-1BB-bindingagents; insect cell systems infected with recombinant virus expressionvectors (for example, baculovirus) containing a nucleic acid encodingimmunogenic stimuli or agonistic 4-1BB-binding agents; plant cellsystems infected with recombinant virus expression vectors (for example,cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) ortransformed with recombinant plasmid expression vectors (for example, Tiplasmid) containing a nucleotide sequence encoding immunogenic stimulior agonistic 4-1BB-binding agents; or mammalian cell systems (forexample, COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38, and NIH 3T3 cells)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (for example, the metallothioneinpromoter) or from mammalian viruses (for example, the adenovirus latepromoter and the vaccinia virus 7.5K promoter). Also useful as hostcells are primary or secondary cells obtained directly from a mammal andtransfected with a plasmid vector or infected with a viral vector.

Cells transfected or transduced with the expression vectors of theinvention can then be used, for example, for large or small scale invitro manufacture of an immunogenic stimulus or agonistic 4-1BB-bindingagent by methods known in the art. In essence, such methods involveculturing the cells under conditions that maximize production of thepolypeptide and isolating the polypeptide from the cells or from theculture medium.

For the methods of the invention, it is often required that theimmunogenic stimuli and/or agonistic 4-1BB-binding agents be purified.Methods for purifying biological macromolecules (e.g., proteins) areknown in the art. The degree of purity of the macromolecules can bemeasured by any appropriate method, e.g., column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis.

A T cell whose response is enhanced, whose anergy is reversed, or inwhich induction of anergy is prevented by the methods of the inventioncan be a CD4+ T cell or a CD8+ T cell. The invention is not limited by:(a) the T cell having any particular phenotype (e.g., CD4+ or CD8+) orfunction (e.g., cytotoxicity, helper activity, immune deviatingactivity, or suppressive activity); or (b) the MHC molecules by whichthe T cell is restricted being of any particular class. While themajority of T cells with cytotoxic activity are CD8+ and recognizepeptide-epitopes bound to MHC class I molecules, CD4+ CTL that recognizeantigenic peptides bound to MHC class II molecules are known in the art.CD4+ CTL that recognize peptides bound to MHC class I molecules and CD8+CTL that recognize antigenic peptides bound to MHC class II moleculeshave also been described. In addition, while the majority of T cellswith helper and/or immune deviating activity are CD4+ T cells andrecognize antigenic peptides bound to MHC class II molecules, theseactivities have also been observed in MHC class I restricted CD8+ Tcells. Similarly, while most immunosuppressive T cells are CD8+ T cells,CD4+ T cells with immunosuppressive activity have also beendemonstrated. The methods of the invention are applicable to all these Tcells. Preferred responses will be those of MHC class I restricted CTLand MHC class II restricted CD4+ helper/immune deviating T cells.Responses of MHC class I restricted CTL are particularly preferred.

The methods of the invention can be performed in vitro or in vivo.

In vitro methods

In vitro applications can be useful, for example, in basic scientificstudies of immune mechanisms or for production of activated T cells foruse in either studies on T cell function or, for example, passiveimmunotherapy. Furthermore, a 4-1BB-binding agent can be added to invitro assays (e.g., in T cell proliferation assays) designed to test forimmunity to an antigen of interest in a subject from which the T cellswere obtained. Addition of a 4-1BB-binding agent to such assays would beexpected to result in a more potent, and therefore more readilydetectable, in vitro response. However, the methods of the inventionwill preferably be in vivo or ex vivo (see below).

In the in vitro methods of the invention, lymphoid cells (consisting ofor including T cells) obtained from a mammalian subject are culturedwith any of the above described immunogenic stimuli and agonistic4-1BB-binding agents. The lymphoid cells can be from a subjectpre-exposed to a relevant antigen (in any of the forms described above);alternatively, the donor of the lymphoid cells need not have beenexposed to the antigen. The cultures can also be supplemented with oneor more cytokines or growth factors such as, without limitation,interleukin-(IL-)1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-12, IL-13, IL-15,interferon-γ (IFN-γ). tumor necrosis factor-α (TNF-α), granulocytemacrophage colony-stimulating factor (GM-CSF), or granulocyte-colonystimulating factor (G-CSF). The cultures can be “restimulated” as oftenas necessary. The cultures can also be monitored at various times toascertain whether the desired level of immune reactivity (e.g., CTL orhelper T cell activity) has been attained.

In Vivo Methods

The methods of the invention are generally useful for enhancing immuneresponses, reversing anergy in T cells, or preventing the induction ofanergy in T cells. Immune responses generated can be prophylactic ortherapeutic. However, the responses generated need have neitherprophylactic nor therapeutic efficacy. They can be used, for example,(a) to produce large numbers of activated T cells for use in basicscientific studies of T cell activity; or (b) to enhance T cellresponses that provide helper activity for antibody-producing B cellsand thereby facilitate the production of large quantities of antibodiesin mammals (e.g., rabbits, goats, sheep, or horses) that aresubsequently isolated from the animals and used for purposes such asantigen detection or purification, or (c) for immunization of animals(e.g., mice, rats, or hamsters) with a view to making monoclonalantibodies.

The methods of the invention can be used, for example, for prophylaxisor therapy against (a) infectious diseases due to any of the infectiousagents listed herein; or (b) cancers such as any of those listed herein.In addition to being useful for the treatment of a wide variety ofcases, in cases where a subject is at relatively high risk for a cancer(e.g., prostate cancer in men over 50 years of age, lung cancer in atobacco smoker, or melanoma in a subject with multiple nevi),appropriate methods can be used for prophylaxis. In regard to infectiousmicroorganisms, the methods can be particularly useful in the preventionand/or therapy of diseases involving intracellular microorganisms (i.e.infectious agents that replicate inside a cell), e.g., viruses such asinfluenza virus or HIV, intracellular bacteria such M. tuberculosis, andintracellular protozoans such as P. falciparum or any of the otherinfectious agents listed herein.

As used herein, “prophylaxis” can mean complete prevention of thesymptoms of a disease, a delay in onset of the symptoms of a disease, ora lessening in the severity of subsequently developed disease symptoms.“Prevention” of a disease should mean that symptoms of the disease(e.g., an infection) are essentially absent. As used herein, “therapy”can mean a complete abolishment of the symptoms of a disease or adecrease in the severity of the symptoms of the disease. As used herein,a “protective” immune response is an immune response that isprophylactic and/or therapeutic.

The methods of the invention can be applied to a wide range of species,e.g., humans, non-human primates (e.g., monkeys), horses, cattle, pigs,sheep, goats, dogs, cats, rabbits, guinea pigs, hamsters, rats, andmice. The immunogenic stimuli and agonistic 4-1BB-binding agents can bederived from any of these species. They will preferably be, but notnecessarily, used to generate immune responses of cells of the speciesfrom which they were derived.

In one in vivo approach, one or more of any of the above the immunogenicstimuli and one or more of any of the above the agonistic 4-1BB-bindingagents (in any of the forms described above) are administered to asubject of any of the above mammalian species. The immunogenic stimulican be administered at the same time as the agonistic 4-1BB-bindingagent(s), or separately, i.e., before or after administration of theagonistic 4-1BB-binding agent(s). Generally, the immunogenic stimuli andthe agonistic 4-1BB-binding agents, whethered administered per se oradministered in the form of recombinant cells either secreting them orexpressing them on their surfaces, will be suspended in apharmaceutically-acceptable carrier (e.g., physiological saline) andadministered orally or transdermally or injected (or infused)intravenously, subcutaneously, intramuscularly, intraperitoneally,intrarectally, intravaginally, intranasally, intragastrically,intratracheally, or intrapulmonarily. They can be delivered directly toan appropriate lymphoid tissue (e.g. spleen, lymph node, ormucosal-associated lymphoid tissue (MALT)). The dosage required dependson the route of administration, the nature of the formulation, thenature of the patient's illness, the subject's size, weight, surfacearea, age, and sex, other drugs being administered, and the judgment ofthe attending physician. Suitable dosages for soluble immunogenicstimuli and soluble agonistic 4-1BB-binding agents are in the range of0.01-100.0 mg/kg. Wide variations in the needed dosage are to beexpected in view of the variety of immunogenic stimuli and agonistic4-1BB-binding agents available and the differing efficiencies of variousroutes of administration. For example, oral administration would beexpected to require higher dosages than administration by intravenousinjection. Variations in these dosage levels can be adjusted usingstandard empirical routines for optimization, as is well understood inthe art. Administrations can be single or multiple (e.g., 2- or 3-, 4-,6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold). Encapsulation of theimmunogenic stimuli and/or agonistic 4-BB-binding agents in a suitabledelivery vehicle (e.g., polymeric microparticles or implantable devices)may increase the efficiency of delivery, particularly for oral delivery.In addition, adjuvants can be used together with the immunogenicstimuli. Suitable adjuvants include cholera toxin (CT), E. coli heatlabile toxin (LT), mutant CT (MCT) [Yamamoto et al. (1997) J. Exp. Med.185:1203-1210] and mutant E. coli heat labile toxin (MLT) (Di Tommaso etal. (1996) Infect. Immunity 64:974-979]. MCT and MLT contain pointmutations that substantially diminish toxicity without substantiallycompromising adjuvant activity relative to that of the parent molecules.Other useful adjuvants include alum, Freund's complete and incompleteadjuvant, and RIBI. In addition, one or more of the above-listedcytokines or growth factors can be administered (by any of the routesrecited herein) to the subject, before, at the same time as, or afteradministration of immunogenic stimuli or agonistic 4-1BB-binding agents.Moreover, where tumor cells, APC, or hybrid cells are used as theimmunogenic stimulus, such cells, in addition to expressing on theirsurface or secreting recombinant agonistic 4-1BB-binding agents, canalso express on their surface or secrete either (a) one or morerecombinant costimulatory molecules (e.g., B7.1, B7.2, B7-H1, B7-H2,B7-H3, or B7-H4) and/or (b) one or more recombinant cytokines orrecombinant growth factors such as those listed above, e.g., GM-CSF.Cells expressing on their surface or secreting the above recombinantmolecules will have been transfected (stably or transiently) ortransformed with one or more nucleic acids (e.g., expression vectors)encoding the molecules.

Alternatively, one or more polynucleotides containing nucleic acidsequences encoding one or more immunogenic stimuli and/or one or moreagonistic 4-1BB-binding agents of interest can be delivered to anappropriate cell of the animal. Expression of the coding sequences willpreferably be directed to lymphoid tissue of the subject by, forexample, delivery of the polynucleotide to the lymphoid tissue. This canbe achieved by, for example, the use of a polymeric, biodegradablemicroparticle or microcapsule delivery vehicle, sized to optimizephagocytosis by phagocytic cells such as macrophages. For example, PLGA(poly-lacto-co-glycolide) microparticles approximately 1-10 μm indiameter can be used. The polynucleotides are encapsulated in thesemicroparticles, which are taken up by macrophages and graduallybiodegraded within the cell, thereby releasing the polynucleotide. Oncereleased, the nucleic acid sequence are expressed within the cell. Asecond type of microparticle is intended not to be taken up directly bycells, but rather to serve primarily as a slow-release reservoir ofnucleic acid that is taken up by cells only upon release from themicro-particle through biodegradation. These polymeric particles shouldtherefore be large enough to preclude phagocytosis (i.e., larger than 5μm and preferably larger than 20 μm).

Another way to achieve uptake of the nucleic acid is using liposomes,prepared by standard methods. The vectors can be incorporated alone intothese delivery vehicles or co-incorporated with tissue-specificantibodies. Alternatively, one can prepare a molecular conjugatecomposed of a plasmid or other vector attached to poly-L-lysine byelectrostatic or covalent forces. Poly-L-lysine binds to a ligand thatcan bind to a receptor on target cells [Cristiano et al. (1995), J. Mol.Med. 73, 479]. Alternatively, lymphoid tissue specific targeting can beachieved by the use of lymphoid tissue-specific transcriptionalregulatory elements (TRE) such as a B lymphocyte, T lymphocyte, ordendritic cell specific TRE. Lymphoid tissue specific TRE are known[Thompson et al. (1992), Mol. Cell. Biol. 12, 1043-1053; Todd et al.(1993), J. Exp. Med. 177, 1663-1674; Penix et al. (1993), J. Exp. Med.178, 1483-1496]. Delivery of “naked DNA” (i.e., without a deliveryvehicle) to an intramuscular, intradermal, or subcutaneous site isanother means to achieve in vivo expression.

In the relevant polynucleotides (e.g., expression vectors) the nucleicacid sequence encoding the relevant immunogenic stimulus (or agonistic4-1BB-binding agent) with an initiator methionine and optionally atargeting sequence (see above), is operatively linked to a promoter orenhancer-promoter combination.

A promoter is a TRE composed of a region of a DNA molecule, typicallywithin 100 basepairs upstream of the point at which transcriptionstarts. Enhancers provide expression specificity in terms of time,location, and level. Unlike a promoter, an enhancer can function whenlocated at variable distances from the transcription site, provided apromoter is present. An enhancer can also be located downstream of thetranscription initiation site. To bring a coding sequence under thecontrol of a promoter, it is necessary to position the translationinitiation site of the translational reading frame of the peptide orpolypeptide between one and about fifty nucleotides downstream (3′) ofthe promoter. The coding sequence of the expression vector isoperatively linked to a transcription terminating region.

Suitable expression vectors include plasmids and viral vectors such asherpes viruses, retroviruses, vaccinia viruses, attenuated vacciniaviruses, canary pox viruses, adenoviruses and adeno-associated viruses,among others.

Polynucleotides can be administered in a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are biologicallycompatible vehicles which are suitable for administration to a human orother mammalian subject, e.g., physiological saline. A therapeuticallyeffective amount is an amount of the polynucleotide that is capable ofproducing a medically desirable result (e.g., a detectable T cellresponse) in a treated animal. As is well known in the medical arts, thedosage for any one patient depends upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Dosages will vary, but apreferred dosage for administration of polynucleotide is fromapproximately 10⁶ to 10¹² copies of the polynucleotide molecule. Thisdose can be repeatedly administered, as needed. Routes and frequency ofadministration can be any of those listed above.

Ex Vivo Methods

In one ex vivo approach, lymphoid cells, including T cells (CD4+ and/orCD8+ T cells), are isolated from a subject and exposed to one or moreimmunogenic stimuli and one or more agonistic 4-1BB-binding agents invitro (see above). Such T cells can be, for example, anergic T cells inwhich it is desired to reverse anergy. The lymphoid cells can be exposedonce or multiply (e.g., 2, 3, 4, 6, 8, or 10 times). The level of immuneactivity (e.g., CTL activity) in the lymphoid cells can be tested afterone or more exposures. Once the desired activity and level of thatactivity is attained, the cells are reintroduced into the subject (oranother subject) via any of the routes listed herein. The therapeutic orprophylactic efficacy of this ex vivo approach is dependent on theability of the ex vivo activated lymphocytes to exert, directly orindirectly, a neutralizing or cytotoxic effect on, for example,infectious microorganisms, host cells infected with microorganisms, ortumor cells.

An alternative ex vivo strategy can involve transfecting or transducingcells obtained from a subject with one or more polynucleotides encodingone or more immunogenic stimuli and one or more agonistic 4-1BB-bindingagents. The transfected or transduced cells are then returned to thesubject or another subject. While such cells would preferably behemopoietic cells (e.g., bone marrow cells, macrophages, monocytes,dendritic cells, or B cells) they could also be any of a wide range oftypes including, without limitation, fibroblasts, epithelial cells,endothelial cells, keratinocytes, or muscle cells in which they act as asource of the one or more immunogenic stimuli and one or more agonistic4-1BB-binding agents for as long as they survive in the subject. The useof hemopoietic cells, that include the above APC, would be particularlyadvantageous in that such cells would be expected to home to, amongothers, lymphoid tissue (e.g., lymph nodes or spleen) and thus theimmunogenic stimuli and agonistic 4-1BB-binding agents would be producedin high concentration at the site where they exert their effect, i.e.,enhancement of an immune response. In addition, if APC expressing atransgene encoding one or more agonistic 4-1BB-binding agents are used,the APC expressing the exogenous can be, but are not necessarily, thesame APC that presents an alloantigen or antigenic peptide to therelevant T cell. The agonistic 4-1BB-binding agents can be secreted bythe APC or expressed on its surface. Prior to administering recombinantAPC to a subject, they can optionally be exposed to the above-listedsources of antigens or antigenic peptides of interest, e.g., those oftumors or infectious microorganisms. The same genetic constructs andtrafficking sequences described for the in vivo approach can be used forthis ex vivo strategy. Furthermore, tumor cells or hybrid cells producedby fusion of APC (e.g., dendritic cells) and tumor cells can betransfected or transformed by one or more vectors encoding one or moreagonistic 4-1BB-binding agents. Such cells, preferably treated with anagent (e.g., ionizing irradiation or mitomycin C) that ablates theirproliferative capacity, are then administered to a subject with therelevant cancer where, due to their expression of the exogenousagonistic 4-1BB-binding agents (on their cell surface or by secretion),they can stimulate enhanced tumoricidal T cell immune responses. It isunderstood that the tumor cells which, after transfection ortransformation with agonistic 4-1BB-binding agent encoding nucleicacids, are administered to a subject with cancer can have been obtainedfrom an individual other than the subject. Similarly, tumor cells usedfor the production of hybrid cells that express recombinant agonistic4-1BB-binding agents and are administered to a subject with cancer, canhave been obtained from an individual other than the subject.

The ex vivo methods can include the steps of harvesting cells (e.g.,tumor cells or APC) from a subject, culturing the cells, transducingthem with one or more expression vectors, and maintaining the cellsunder conditions suitable for expression of the immunogenic stimuliand/or agonistic 4-1BB-binding agents. These methods are known in theart of molecular biology. The transduction step is accomplished by anystandard means used for ex vivo gene therapy, including calciumphosphate, lipofection, electroporation, viral infection, and biolisticgene transfer. Alternatively, liposomes or polymeric microparticles canbe used. Cells that have been successfully transduced are then selected,for example, for expression of the coding sequence or of a drugresistance gene. The cells may then be lethally irradiated (if desired)and injected or implanted into the patient. The methods can include theadditional step of making the above-described cells hybrids that areinjected or implanted into the patient.

It is understood that the methods of invention can involve combinationsof the above in vivo and ex vivo approaches. Thus, for example, animmunogenic stimulus can be provided in the form of a peptide-epitopeand agonistic 4-1BB-binding agent in the form of either a nucleic acidencoding it or cells transformed with a nucleic acid encoding it.

The methods of the invention can be applied to any of the diseases andspecies listed here. Methods to test whether a given modality istherapeutic for or prophylactic against a particular disease are knownin the art. Where a therapeutic effect is being tested, a testpopulation displaying symptoms of the disease (e.g., cancer patients) istreated by a method of the invention, using any of the above describedstrategies. A control population, also displaying symptoms of thedisease, is treated, using the same methodology, with a placebo.Disappearance or a decrease of the disease symptoms in the test subjectswould indicate that the method was therapeutic.

By applying the same strategies to subjects prior to onset of diseasesymptoms (e.g., presymptomatic subjects considered to likely candidatesfor cancer development (see above) or experimental animals in which anappropriate disease spontaneously arises or can be deliberately induced,e.g., multiple murine cancers, the method can be tested for prophylacticefficacy. In this situation, prevention of onset of disease symptoms istested. Analogous strategies can be used to test for the efficacy of themethods in the prophylaxis of a wide variety of infectious diseases,e.g., those involving any of the microorganisms listed above.

4-1BB-specific Antibodies

The invention features antibodies that bind specifically to human andmouse 4-1BB. Such antibodies can be polyclonal antibodies present in theserum or plasma of animals (e.g., mice, rabbits, rats, guinea pigs,sheep, horses, goats, cows, or pigs) that have been immunized with therelevant 4-1BB polypeptide (or a peptide fragment thereof) usingmethods, and optionally adjuvants, known in the art. Such polyclonalantibodies can be isolated from, for example, serum, plasma, or ascitesby methods known in the art. Monoclonal antibodies (mAb) that bind to4-1BB polypeptides or fragments are also encompassed by the invention.These mAbs include those produced by the 2A, 5.9, and 5.10 hybridomas(see Examples).

Methods of making and screening monoclonal antibodies are well known inthe art. Once the desired antibody-producing hybridoma has been selectedand cloned, the resulting antibody can be produced by a number of invivo and in vitro methods known in the art. For example, the hybridomacan be cultured in vitro in a suitable medium for a suitable length oftime, followed by the recovery of the desired antibody from thesupernatant. The length of time and medium are known or can be readilydetermined.

Additionally, recombinant antibodies specific for 4-1BB, such aschimeric and humanized monoclonal antibodies comprising both human andnon-human portions, are within the scope of the invention. Such chimericand humanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example, using methods described inRobinson et al., International Patent Publication PCT/US86/02269; Akiraet al., European Patent Application 184,187; Taniguchi, European PatentApplication 171,496; Morrison et al., European Patent Application173,494; Neuberger et al., PCT Application WO 86/01533; Cabilly et al.,U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application125,023; Better et al. (1988) Science 240:104143; Liu et al. (1987) J.Immunol. 139:3521-26; Sun et al. (1987) PNAS 84:214-18; Nishimura et al.(1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-49;Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-59; Morrison, (1985)Science 229:1202-07; Oi et al. (1986) BioTechniques 4:214; Winter, U.S.Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-25; Veroeyan etal. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-60.

Also included within the scope of the invention are antibody fragmentsand derivatives which contain at least the functional portion of theantigen binding domain of an antibody that binds specifically to 4-1BB.Antibody fragments that contain the binding domain of the molecule canbe generated by known techniques. For example, such fragments include,but are not limited to: F(ab′)₂ fragments that can be produced by pepsindigestion of antibody molecules; Fab fragments that can be generated byreducing the disulfide bridges of F(ab′)₂ fragments; and Fab fragmentsthat can be generated by treating antibody molecules with papain and areducing agent. See, e.g., National Institutes of Health, 1 CurrentProtocols In Immunology, Coligan et al., ed. 2.8, 2.10 (WileyInterscience, 1991). Antibody fragments also include Fv (e.g., singlechain Fv (scFv)) fragments, i.e., antibody products in which there arefew or no constant region amino acid residues. An scFv fragment is asingle polypeptide chain that includes both the heavy and light chainvariable regions of the antibody from which the ScFv is derived. Suchfragments can be produced, for example, as described in U.S. Pat. No.4,642,334, which is incorporated herein by reference in its entirety.

The following Examples are meant to illustrate, not limit, theinvention.

EXAMPLES Example 1 Materials and Methods

Tumor models and peptides C3 cells, generated fromHPV-16-/EJras-transformed C57BL/6 (B6) mouse embryo cells [Feltkamp etal. (1993) Eur. J. Immunol. 23:2242-2249], were a gift from Dr. W.Martin Kast (Loyola University, Chicago, Ill.). A line of EL4 cells(EL4E7) transfected with cDNA encoding the human papilloma virus-16(HPV-16) E7 polypeptide [Tindle et al. (1995) Clin. Exp. Immunol.101:265-271] was a gift from Dr. Germain J. P. Fernando (University ofQueensland, Brisbane, Australia). The TC-1 cell line [Liu et al. (1996)Cancer Res. 56:21-6] was a gift from Dr. T. C. Wu (Johns HopkinsUniversity, Baltimore, Md.) and the B16-F10 melanoma line [Dranoff etal. (1993) Proc. Natl. Acad. Sci. USA 90:3539-3543] was a gift from Dr.Glenn Dranoff (Dana-Farber Cancer Institute, Boston, Mass.). The EL4,RMA-S and S49.1 murine T cell lymphoma lives were of B6 origin and werepurchased from the American Type Culture Collection (Manassas, Va.). Theregressor P815 mastocytoma (P815R), which has been previously described[Nieland et al. (1999) J. Cell Biochem 73:145-152], was obtained fromDr. W. Martin Kast, Loyola University, Chicago, Ill. All cell lines weremaintained in a complete tissue culture medium of RPMI 1640 (LifeTechnologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum(FBS) (HyClone, Logan, Utah), 25 mM HEPES, 2 mM glutamine, 100 U/mlpenicillin G and 100 μg/ml streptomycin sulfate.

The E7 peptide (RAHYNIVTF) (SEQ ID NO:7) contained the minimalH-2D^(b)-restricted CTL epitope [Feltkamp et al. (1993) Eur. J. Immunol.23:2242-2249] of HPV-16 E7 protein. The trp-2 peptide (SVYDFFVWL) (SEQID NO:8) is a H-2K^(b)-restricted epitope first identified in the B16melanoma [Bloom et al. (1997) J. Exp. Med. 185:453-459; Schreurs et al.(2000) Cancer Res. 60:6995-7001]. The Vp2 control peptide (FHAGSLLVPM)(SEQ ID NO:9) contains an H-2D^(b)-restricted CTL epitope derived fromthe Theiler's Murine Encephalomyelitis Virus [Johnson et al. (1999).J.Virol. 73:3702-3708]. The OVA (257-264) peptide (SIINFEKL) (SEQ IDNO:10) (referred to in the OT-1 T cell anergy experiments described inExamples 10-13 as the “OVA peptide”) is a H-2Kb-restricted CTL epitopederived from chicken ovalbumin [Curtsinger et al. (1998) J. Immunol.160:3236-3243; Moore et al. (1988) Cell 54:777-785]. The OVA (55-62)peptide (KVVRFDKL) (SEQ ID NO: 11) used as a “control peptide” in theOT-1 T cell anergy experiments described in Examples 10-13 is aH-2K^(b)-restricted CTL epitope from chicken ovalbumin [El-Shami et al.(1999) Eur. J. Immunol. 29:3295-3301] The P1A (35-43) peptide(LPYLGWLVF) (SEQ ID NO: 12) is an H-2L^(d) restricted CTL epitope. Allpeptides were synthesized by the Mayo Molecular Biology Core Facilityand the purity of the peptides was >90% by reverse-phase HPLCpurification. The peptides were dissolved in dimethyl sulfoxide (DMSO)and reconstituted in phosphate buffered saline (PBS) to a finalconcentration of 1 mg/ml (5% DMSO) for administration to mice.

Female B6 mice were purchased from the National Cancer Institute(Frederick, Md.). Age-matched mice, 6-10 weeks old, were used for allexperiments. Tumor cells in 0.1 ml of PBS were injected subcutaneously(s.c.) into the right shaved flanks. Mice were given 1×10⁶ C3 cells or4×10⁶ EL4E7 cells. Tumor size (the average of two perpendiculardiameters in mm) was measured weekly as previously described [Tamada etal. (2000) Nature Med. 6:283-289]. For lung metastases models, 1×10⁴TC-1 or 1×10⁵ B16-F10 cells were injected in 0.5 ml of Hank's BufferedSalt Solution into the tail vein of mice. Mice bearing subcutaneoustumors were immunized intradermally (i.d.) at a site contralateral tothe tumor with 50 μg of peptide emulsified in incomplete Freund'sadjuvant (IFA) (Sigma Chemicals, St. Louis, Mo.). Mice bearing lungmetastases were immunized bilaterally i.d. with a total of 100 μg ofpeptide emulsified in IFA. Antibodies administered to mice were injectedintraperitoneally (i.p.) in 0.5 ml of PBS.

For experiments involving challenge with P815R tumor cells, 1.5×10⁴P815R cells were injected intradermally into the shaved flanks of DBA/2mice.

OT-1 transgenic mice, which have been previously described [Strome etal. (2002) Cancer Research 62:1884-1889], were obtained from Dr. LarryPease, Mayo Clinic, Rochester, Minn. All the CD8+ T cells of the OT-1mice express an antigen specific T cell receptor (TCR) specific for theOVA (257-264) peptide bound to the murine H-2K^(b) MHC class I molecule.

Antibodies, tetramers, and fusion protein To prepare a 4-1BBIg fusionprotein, cDNA encoding the extracellular domain of mouse 4-1BB wasamplified from cDNA produced from RNA isolated from concanavilinA-activated spleen cells using sequence specific primers and was fusedto the CH₂-CH₃ domain of mouse IgG2a in the expression plasmid pmIgV[Chapoval et al. (2000) Nature Med. 6:283-289]. The resulting expressionvector was transfected into CHO cells. The protein in the culturesupernatants of a transfected clone was purified using a HiTrap ProteinG-Sepharose column (Amersham Pharmacia Biotech, Piscataway, N.J.) anddialyzed into lipopolysaccharide-free PBS.

A rat monoclonal antibody (mAb) against 4-1BB was generated byimmunizing a Lewis rat (Harlan Sprague Dawley, Indianapolis, Ind.) withmouse 4-1BBIg. Hybridomas were produced by fusing rat spleen cells withmouse Sp2/0 myeloma cells and the culture supernatants were screened byELISA. The hybridoma secreting the mAb 2A was selected for furtherexperiments. Hybridoma 2A was grown in RPMI 1640 supplemented with 10%low IgG FBS (Life Technologies) and 25 mM HEPES and supernatant washarvested and concentrated using a tangential flow miniplateconcentrator (Millipore, Bedford, Mass.). The 2A mAb was purified fromthe concentrated supernatant using a 5 ml HiTrap Protein G-Sepharosecolumn (Amersham Pharmacia Biotech, Piscataway, N.J.). The purified mAbwas dialyzed against PBS and concentrated using a Centriprepconcentrator (Millipore, Bedford, Mass.). The isotype of the 2A mAb wasdetermined using biotinylated, isotype-specific antibodies (CaltagLaboratories, Burlingame, Calif.). It was found to be an IgG2a antibodywith kappa light chains.

Purified mAb specific for mouse CD3, CD28, 4-1BB, fluoresceinisothiocyanate-(FITC-) conjugated CD8, CD69, CD25, CD49A, and isotypecontrol mAb, and Cy-Chrome-conjugated CD8 mAb were purchased fromPharMingen (San Diego, Calif.). The FITC-conjugated goat anti-rat IgGantibody was purchased from Biosource International (Camarillo, Calif.).Rat IgG antibodies (Sigma Chemical, Gilbertsville, Pa.) were used ascontrols.

Tetramers of the mouse H-2D^(b) major histocompatibility complex (MHC)class I molecule bound to either the E7 peptide (H-2D^(b)-E7) or thecontrol Vp2 peptide (H-2D^(b)-Vp2) were prepared as previously described[Johnson et al. (1999) J. Virol. 73:3702-3708]. Briefly, H-2D^(b)α-chain and human β₂-microglobulin were isolated from a bacterialexpression system and subsequently folded in the presence of excesspeptide. The folded monomeric complexes were desalted and biotinylated.Following cation exchange purification, the monomeric complexes wereconjugated to strepavidin labeled with the fluorescent dye, phycoerythin(PE), thereby forming fluorescent tetrameric complexes. The PE-labeledtetramers generated were then purified by size exclusion gel filtration.

A tetramer composed of four mouse H-2K^(b) molecules bound to the OVA(257-264) peptide and labeled with PE (sometimes referred to below asthe “OVA tetramer”) was obtained from the NIH tetramer core facility(Atlanta, Ga.).

T cell costimulation assay The method used to assay costimulatoryactivity of mAb was described previously [Tamada et al. (2000) NatureMed. 6:283-289]. Briefly, nylon wool (NW)-purified mouse splenic T cells(2.5×10⁶/ml) were added to 96-well plates which had been coated with amAb against CD3 (0.1 μg/ml) and the indicated concentrations of rat IgGor mAb 2A. The proliferation of T cells was assessed by the addition of1 μCi/well of [³H]-thymidine (³H-TdR) to the 3-day cultures 15 hoursbefore harvesting of the cultures onto fiber glasstilters. ³H-TdRincorporated into the T cells was measured in a MicroBeta TriLux liquidscintillation counter (Wallac, Turku, Finland).

FACS analysis and sorting T cells were positively selected using FITCconjugated mAbs against CD4 and CD8, metal microbeads coated withantibody specific for FITC, and a magnet as instructed by themanufacturer (Miltenyi Biotec, Auburn, Calif.). The purity of theisolated T cells was routinely greater than 95%, as assessed by flowcytometry using a mAb against CD3. Purified T cells (2.5×10⁶ cells/ml)from mouse spleens were stimulated in the wells of 24-well tissueculture plates coated with mAbs against CD3 (5 μg/ml) and CD28 (1μg/ml). After 24 hours, T cells were collected and stained for 30 min at4° C. with 1 μg mAb 2A, either alone or in the presence of 4-1BBIg (2μg/ml), in 50 μl PBS supplemented with 3% FBS and 0.02% azide. The cellswere washed and incubated an additional 30 min at 4° C. withFITC-conjugated goat antibody specific for rat IgG. After washing thecells were fixed in 1% paraformaldehyde and fluorescence was analyzedwith a FACS (Becton Dickinson, Mountain View, Calif.). S49.1 cells werestained in a similar fashion. In brief, 1×10⁶ S49.1 cells were stainedwith either mAb 2A or the mAb specific for 4-1BB purchased fromPharmingen (clone 1AH2). After washing, the cells were stained with aFITC-conjugated antibody specific for rat IgG, washed, fixed andanalyzed.

Tumor-draining lymph nodes (TDLN) from immunized mice were harvested onday 7 and stained with PE-labeled H-2D^(b)E7 or H-2D^(b)-Vp2 tetramericcomplexes and FITC-conjugated CD8 as previously described [Johnson etal. (1999) J. Virol. 73:3702-3708]. Five×10⁶ TDLN cells were incubatedwith 2.5×10⁵ UV-irradiated C3 cells for 4 days. Cells were subsequentlystained with the PE-labeled tetramers and FITC-conjugated CD8 Afterextensive washing, cells were re-suspended in PBS with 750 ng/mlpropidium iodide. Gates were drawn to include viable CD8⁺ cells only.

FACS analysis for the OT-1 T cell anergy experiments was peformedessentially as described above but using the staining reagents indicatedbelow.

For FACS sorting of OT-1 TCR expressing T cells, T cells were purifiedfrom spleens and lymph nodes of mice using microbead coated withantibody specific for Thy1.2 according to the manufacturers instructions(Miltenyi Biotec, San Diego, Calif.). Purified Thy1.2+ cells weresubsequently stained with the OVA tetramer described above. Positivelystained cells were sorted using a FACSVantage Flow Cytometry System (B DImmunocytometry Systems, San Jose, Calif.). At least 90% of the sortedcells were both OVA tetramer positive and CD8+.

Assay for CTL activity In the experiments on the C3 tumor, effectorcells were obtained by co-culturing draining LN cells with irradiated C3cells for 4 days. Effector cells were harvested from the cultures andtested for CTL activity using a standard 4-hr⁵¹Cr release assay withtumor cell targets at the indicated effector to target cell (E:T)ratios. Peptide-pulsed target cells were generated by culturing thetarget cells with 10 μg/ml of the peptide at 28° C. for 18 hours priorto use. The CTL assay used for the OT-1 T cell anergy experiments wasperformed similarly using the target cells indicated below.

Preparation of murine DC Murine DC were prepared from bone marrow. Micewere sacrificed and dipped in 70% ethanol (EtOH). After removing excessEtOH, the hind limbs were exposed and the hip joint dislocated. Muscleparenchyma was removed and the bones placed briefly in 70% EtOH and thenin complete medium (CM; RPMI 1640, 10% heat inactivated FBS (HycloneLaboratories, Inc., Logan, Utah), Fungizone (0.5 μg/ml), β-ME (2×10⁻⁵M),sodium pyruvate (1 mM), non-essential amino acids (0.1 mM), penicillinand streptomycin (100 μg/ml), glutamine (2 mM) and Gentamycin (50μg/ml)). Both ends of the bones were cut to expose the marrow and a 3 ccsyringe (filled with CM) with a 25 gauge needle was used to eject themarrow into a 10 mm cell culture dish, containing CM. Following marrowextraction, the cells were separated from stromal components bystraining through a steel sieve and, after pelleting by centrifugation,were resuspended in 1.0 ml medium of containing 10 μg/ml of anti-classII (I-A^(b)) mAb, anti-Mac 3 mAb, anti-CD8a mAb (HO2.2), anti-CD45R(B220) mAb, anti-CD3e mAb, and anti-GR-1 mAb (all from PharMingen, Inc.San Diego, Calif.), and incubated on ice for 20 minutes. The cells werethen washed once and resuspended in rabbit serum (diluted 1:30 inmedium) (Cedarlane Laboratories, Ltd., Hornby, Ontario, Canada) as asource of lytic complement at a concentration of 10⁷ cells/ml andincubated at 37° C. for 45 minutes. The cells were then washed, platedin 100 mm cell culture dishes at a concentration of 10⁷ cells in 10 mlof CM supplemented with 10 ng/ml granulocyte macrophage-colonystimulating factor (GM-CSF) and 1 ng/ml interleukin-4 (IL-4), andcultured at 37° C. Non-adherent cells were removed from the cultures onday 2 and discarded and the cultures containing highly purified DC wereharvested on day 5.

Induction of T cell anergy 3−7×10⁶ lymph node and spleen cells from OT-1mice were injected intravenously (i.v.; tail vein) into wild type B6mice in 0.5 ml Hanks balanced salt solution (HBSS) (Cellgro, Herndon,Va.). 12-24 hours later, experimental mice were given 0.5 mg of OVA(257-264) peptide i.v. in 0.5 ml total volume, while control mice weregiven OVA (55-62) peptide (or PBS alone in some experiments) in asimilar fashion. On the day of peptide administration, and again 3 dayslater, mice were given 100 μg of either rat IgG or anti-CD 137 mAbintraperitoneally (i.p.). Mice were sacrificed at various time pointsfollowing peptide administration and the total number of OT-1 cellspresent in the spleen and lymph nodes of each mouse was determined byOVA tetramer staining. For restimulation experiments, ten days followingthe administration of peptide, spleens were harvested from the mice.After lysing red blood cells in Ack lysis buffer, the spleen cells wereresuspended in RPMI tissue culture medium supplemented as describedabove for the medium used to culture tumo cell lines and plated intriplicate into the wells of a 96-well plate at a density of 5.5×10⁵cells per well in final volume of 200 μl per well. One group of cellswas unstimulated while a second group was restimulated with 1 ng/ml ofOVA peptide. The same day that splenocytes were restimulated, thefrequency of OVA-specific T cells was determined using the H-2K^(b)-OVA(257-264) tetramer. Thus, the absolute number of OT-1 cells added toeach well could be calculated prior to restimulation in vitro.Supernatants were collected from the wells in each group 48 and 72 hoursafter restimulation and IL-2 (48 hr) and IFN-γ (72 hr) production wasmeasured by sandwich ELISA following the manufacturer's instructions(PharMingen, San Diego, Calif.). The proliferation of T cells wasassessed by the addition of 1 μCi/well [³H]-thymidine during the last 15hours of the 3-day culture. [³H]-thymidine incorporation was measured ina MicroBeta TriLux liquid scintillation counter (Wallac, Turku,Finland). Antigen specific proliferation or cytokine production per OT-1cell was calculated by subtracting any nonspecific proliferation (orcytokine production) observed in the unstimulated groups from theproliferation (or cytokine production) observed in the peptidestimulated groups. This was then divided by the number of OT-1 cells(10³) initially present in the well prior to restimulation to derive thenet change in cpm (Δcpm) per 10³ OT-1 cells.

To measure the ability of anti-CD 137 to reverse anergy, OT-1 cells wereadoptively transferred into wild type recipients. Anergy was induced bythe intravenous administration of 0.5 mg OVA peptide as described above.Alternatively, mice received only control peptide or PBS and wereconsidered “naïve” at the time of rechallenge with the antigen. Ten dayslater mice were given 0.5 mg OVA peptide or control peptideintravenously. Mice received 100 μg of either rat IgG or anti-CD 137.Mice were sacrificed at various time points following rechallenge withthe OVA peptide and the number of OT-1 cells present in the spleen andlymph nodes of each mouse was determined by tetramer analysis, asbefore.

Example 2 The Anti-4-1BB mAb 2A and its in vivo Antitumor Effect onEL4E7 Lymphoma and C3 Epithelioma

The specificity of anti-4-1BB mAb was examined. Monoclonal antibody 2Astained >80% of purified T cells that had been activated for 24 hours byanti-CD3 and anti-CD28 mAbs. Binding of the antibody was specific as itcould be competitively inhibited by inclusion of mouse 4-1BBIg in thestaining reaction (FIG. 1A) while inclusion of a control rat IgGantibody did not inhibit binding (data not shown). Furthermore, mAb 2Abinds specifically to the mouse T cell lymphoma S49.1 thatconstitutively expresses 4-1BB (as demonstrated by staining with 1AH2, acommercially available anti-4-1BB mAb (FIG. 1B)). Immobilized mAb 2Aalso enhanced T cell proliferation in a dose-dependent fashion in thepresence of a suboptimal dose of anti-CD3 mAb (FIG. 1C). Therefore, 2Ais a costimulatory mAb similar to others previously described [Melero etal. (1997) Nature Med. 3:682-685; Shuford et al. (1997) 186:47-55].

To test whether mAb 2A induces the regression of established tumors, twomouse tumors were selected. EL4E7 is a thymoma transfected to expressthe HPV-16 E7 gene and C3 is an embryonic epithelial cell linetransformed with HPV-16 and the ras oncogene. Since both tumor linesexpress the E7 gene of HPV-16, CTL responses to the E7 gene product canbe monitored. To determine the antitumor effect of mAb 2A, groups ofmice bearing established EL4E7 or C3 tumors were injected i.p. with 2A(100 μg) at days 7 and 10. As shown in FIG. 2, established EL4E7 tumorsregressed rapidly in the mice injected with 2A while tumors grewprogressively in the mice treated with a control rat IgG antibody (FIG.2). Remarkably, EL4E7 tumors up to 12 mm in diameter regressed within 7days following treatment. In sharp contrast, treatment of mice bearingC3 tumors less than 4 mm in diameter had only a marginal effect. Asshown in a representative experiment (FIG. 2), retardation of tumorgrowth occurred in one out of five mice; tumors in the other four micegrew progressively. The results indicate that, although anti-4-1BB mAberadicates established EL4E7 tumors, C3 tumors are refractory totreatment. This resistance to treatment is unlikely due to the size ofthe tumor or to any antigenic disparity between the tumors.

Example 3 Presence of CTL that are Ignorant of the E7 Antigens inC3-bearing Mice is Associated with Resistance to mAb 2A Treatment

To understand the mechanisms underlying the resistance of C3 tumors to2A mAb treatment, the activation status of tumor-specific CTL in micebearing C3 tumors was examined. Mice were first inoculatedsubcutaneously (s.c.) with C3 cells and subsequently (3-7 days later)treated with mAb 2A or control rat IgG. Seven days later, TDLN wereharvested, re-stimulated in vitro with irradiated C3 cells, and the CTLactivity of cells harvested from cultures was tested in a standard ⁵¹Crrelease assay. As shown in FIG. 3A, neither EL4E7 nor C3 were lysed bythe in vitro activated TDLN in C3-bearing mice, even if the mice fromwhich the TDLN were obtained had been treated with mAb 2A. It isunlikely that the failure to detect CTL activity was due toinsensitivity of the assay as CTL activity could not be detected againstRMA-S cells pulsed with the E7 peptide or EL4E7 cells, both of which arehighly sensitive target cells. In sharp contrast, E7-specific CTLactivity is routinely detected in TDLN isolated from ELAE7-bearing mice;in addition, this E7-specific CTL activity is enhanced by treatment with2A mAb (FIG. 3B). What would appear to be non-specific lysis of the wildtype RMA-S cells in this assay may be explained by the recentobservation that EL4 and RMA-S cells share a tumor antigen [Van Hall etal. (2000) J. Immunol. 165:869-877].

The frequency of E7 (49-57) specific T cells in C3 TDLN was determinedby double staining with FITC conjugated anti-CD8 mAb and PE-labeled E7tetramer. Consistent with the findings on CTL activity, less than 0.1%of CD8⁺ T cells in TDLN from C3-bearing mice were E7-specific, evenafter in vitro re-stimulation with irradiated C3 cells. This valuerepresents a threshold of “undetected CTL” in the assay since similarresults were also obtained using cells from naïve mice. Furthermore,treatment with the 2A mAb failed to expand E7-specific CTL in C3 TDLN(FIG. 4A, B). In contrast, ˜1% of CD8⁺ cells were E7-specific in thedraining LN of EL4E7-bearing mice treated with the control antibodyafter restimulation with irradiated C3 cells. Treatment with mAb 2A invivo promoted the expansion of E7-specific CTL, as demonstrated by a4-fold increase in the frequency of E7-specific T cells (FIG. 4B). Thefrequency of E7-specific CTL thus to correlated with CTL activity. Moreimportantly, the results indicate that the absence of active E7 specificCTL, rather than suppressed cytolytic activity of specific CTL, in TDLNof C3 bearing mice is responsible for the inability of 2A mAb to boost Tcell responses.

To exclude the possibility that E7-specific CTL are deleted inC3-bearing mice, E7-specific CTL activity was examined in the mice afterimmunization with the E7 (49-57) peptide that contains a H-2D^(b)restricted CTL epitope. Seven days after peptide immunization, drainingLN were harvested, restimulated with irradiated C3 cells and thefrequency of E7 specific CD8⁺ T cells was determined using thefluorescent E7 tetramer. Immunization with the E7 peptide caused asignificant increase of cells that bound the E7 tetramer. Such cellswere not detectable after immunization with a control Vp2 peptide.Treatment with mAb 2A resulted in a further increase in the frequency ofE7-specific CTL (FIG. 5A). Similar results were obtained by immunizationof naïve mice (FIG. 5B). Therefore, E7-specific CTL are present inC3-bearing mice, but are neither activated nor deleted by the C3 cells.Thus E7-specific CTL ignore antigens presented by the C3 tumor. Inaddition, anti-4-1BB mAb alone is unable to break this ignorant state.

Example 4 Regression of Established C3 Epithelioma by Breaking CTLIgnorance with E7 Epitope Peptide and Anti-4-1BB mAb 2A

As shown in Example 3, immunization with the E7 epitope peptideincreases CTL frequency in C3-bearing mice. An experiment was designedto test whether immunization with the E7 peptide is an effectivetreatment for established C3 tumors. Mice bearing C3 tumors for 7 dayswere immunized with either E7 or the control Vp2 peptide and the micewere observed for at least 12 weeks following treatment. They weresacrificed after the tumor reached 15 mm in diameter. Tumor regressionwas observed in only 1 of 11 (9%) mice treated with the E7 peptide only.Therefore, E7 peptide immunization is not sufficient to treatestablished C3 tumors. However, tumors completely regressed in 11 of 15(73%) mice that had received a combined treatment with costimulatory4-1BB mAb plus E7 peptide (COPP). In addition, those tumors that failedto regress in mice after COPP treatment grew more slowly when comparedto tumors in the control groups (FIG. 6). Only 2 of 10 (20%) controlmice treated with mAb 2A and the control Vp2 peptide were tumor free.The mice treated with the control Vp2 peptide and rat IgG antibodyreached 15 mm in diameter within 8 weeks, with the exception of a singlemouse bearing a smaller tumor (<15 mm) for more than 12 weeks (FIG. 6).Therefore, COPP treatment effectively induced the regression ofestablished C3 tumors.

Whether COPP treatment is also effective in treating larger C3 tumorswas determined. As summarized in Table 1, mice bearing C3 tumors for 14days (5-8 mm in diameter at the time of treatment) were treated withCOPP. Tumor regression was observed in 16 of 38 (42%) mice. In contrast,tumor regression was observed in 0%, 9% and 0%, respectively, in micetreated with E7 peptide, mAb 2A alone or the control Ig and Vp2 peptide.Tumors that failed to completely regress in the mice given COPPtreatment were also significantly smaller 21 days following treatmentthan tumors observed in all three control groups of mice.

TABLE 1 Treatment of mice bearing large C3 tumors. Treatment^(a) TumorFree/ Mean Tumor Antibody Peptide Total (%) Diameter (mm)^(b)P-value^(c) 2A E7(49-57) 16/38 (42%)  7.6 +/− 2.4 — Control Ig E7(49-57) 0/11 (0%) 10.5 +/− 3.0 0.017 2A Vp2(121-130)  2/23 (9%) 11.4 +/− 4.00.004 Control Ig Vp2(121-130)  0/8 (0%) 11.5 +/− 3.5 0.005 ^(a)Mice wereinjected with 1 × 10⁶ C3 cells. Two weeks later, mice were immunizedwith the indicated peptide. On the day of immunization and 3 days later,mice were given 100 μg of mAb 2A or a control rat IgG i.p. Tumor sizewas assessed weekly. Data shown was pooled from several experiments.^(b)21 days following treatment, the mean tumor diameter was calculatedfor those tumors which had failed to completely regress. ^(c)TheUnpaired Student's T-test was used to calculate p-values comparing themean tumor diameter of the treatment group which received both the E7peptide and mAb 2A with those of the control groups.

Example 5 Effect of COPP Treatment in Metastasis Models of TC-1 LungCancer and B16-F10 Melanoma

To determine the effect of COPP treatment more vigorously in other,poorly immunogenic tumors, two tumor models were tested. The TC-1 tumorline is derived from primary lung epithelial cells co-transformed withboth the HPV-16 E6, HPV-16 E7 and ras oncogenes [Liu et al. (1996)Cancer Res. 56:21-6]. Therefore, the E7 peptide could be used as animmunogen. B16-F10 is a highly metastatic melanoma line, which presentsthe H-2K^(b)-restricted trp-2 peptide [Dranoff et al. (1993) Proc. Natl.Acad. Sci. USA 90:3539-3543; Bloom et al. (1997) J. Exp. Med.185:453-459; Schreurs et al. (2000) Cancer Res. 60:6995-7001]. B6 micewere injected intravenously (i.v.) with 10⁴ TC-1 cells to establish lungmetastases. Three days after tumor injection, the mice were injecteds.c. with the E7 peptide and i.p. with the 2A mAb (COPP). As wasobserved with the C3 tumor, the administration of mAb 2A alone wasinsufficient to prolong survival in the tumor-bearing mice, as all ofthe mice died within 20 days. E7 peptide immunization alone did prolongsurvival, although all of the mice were dead by day 35. COPP treatmentled to a significant survival advantage in that all of the mice whichhad received both the E7 peptide and mAb 2A survived at least 35 days,the time by which all the mice in the control groups had died (FIG. 7A).Twenty percent of the mice receiving both the E7 peptide and mAb 2A(COPP) were long-term survivors. In a second model, mice were injectedi.v. with 10⁵ B16-F10 cells and treated with COPP three days later. Asbefore, treatment by either mAb 2A or the trp-2 peptide alone wasineffective. However, COPP treatment (trp-2 peptide and mAb 2A) led to asignificant survival advantage for all the mice and long-term survival(>90 days) in 20% of treated mice. Therefore, combined treatment with anantigenic, MHC class I restricted peptide and anti-4-1BB mAb (COPP) maybe therapeutic for established, poorly immunogenic tumors.

Example 6 MAb 2A Enhances the Therapeutic Effect of a Dendritic CellVaccine

In order to determine whether an agonistic mAb specific for murine 4-1BB(mAB 2A) enhances the anti-tumor immune response stimulated by DC-basedvaccines, a murine model of squamous cell carcinoma of the head and neckwas exploited. A poorly immunogenic murine (B6) squamous cell carcinomaline (SCCVII) was used for these experiments. DC were prepared asdescribed above. The DC were “primed” with tumor antigen by culturing DC(5×10⁶ per well) with irradiated SCCVII cells (1×10⁶ well) in the wellsof 24-well tissue culture plates for 24 hours. Four groups of five micewere injected s.c. (in the flank) with 2×10⁵ SCCVII cells. Four daysfollowing injection of the tumor cells, the four groups of animals wereinjected s.c. in the contralateral flank with the following testvaccines:

Group 1: DCs primed with irradiated SCCVII (1×10⁶ DC and 3×10⁶irradiated SCCVII) and Rat IgG (100 μg)

Group 2: DCs primed with irradiated SCCVII (1×10⁶ DC and 3×10⁶irradiated SCCVII) and 2A mAb (100 μg)

Group 3: Rat IgG (100 μg) alone

Group 4: 2A mAb alone (100 μg) alone.

The mice received the test vaccines twice, the second vaccination beinga week after the first. In addition to being administered as a componentof the vaccine, the 2A mAb (or control rat IgG) was administered threedays after each test vaccination. Tumor growth was measured in a blindedfashion to determine therapeutic efficacy. As shown in FIG. 8, 80% ofanimals treated with rat IgG alone developed tumors (group 3; FIG. 8 topright panel). In animals treated with tumor “primed” C and rat IgG(group 1; FIG. 8, bottom right panel) or mAb 2A alone (group 4; FIG. 8,top left panel) there was therapeutic efficacy in 3 out of 5 mice.However, the most pronounced therapeutic effects were observed inanimals treated with tumor “primed” DC and mAb 2A (group 2; FIG. 8,bottom left panel) in which all mice showed tumor regression. These dataclearly demonstrate that antibody specific for 4-1BB is synergistic withtumor “primed” DC vaccines in stimulating therapeutic anti-tumorimmunity.

Example 7 Development of Mouse Anti-human 4-1BB MAbs

In order to generate agonistic mAbs specific for human 4-1BB, a strategysimilar to that described above for generation of mAbs specific formurine 4-1BB was used. A soluble 4-1BB fusion protein composed of anextracellular fragment of human 4-1BB and the CH3-CH3 domain of humanIgG1 was engineered. Mice immunized with this fusion protein producedpolyclonal antibodies to 4-1BB. Spleens from these animals were fusedwith SP2/0 mouse myelomas to produce hybridomas that were screened byfluorescence flow cytometry with 293 cells transfected with cDNAencoding human 4-1BB. Two hybridomas (5.9 and 5.10) producing IgG1 mAbsshowed human 4-1BB-specific staining.

Example 8 T Cell Costimulatory Ability of Mouse anti-Human 4-1BB MAbs

In order to determine whether the two anti-human 4-1BB mAbs described inExample 7 have the ability costimulate a T cell response, a standard invitro costimulation assay was performed. Briefly, purified human T-cellswere cultured with (a) antibody specific for human CD3 coated onto thewell-bottoms of 96-well tissue culture plates at various concentrationsand (b) varying concentrations of the 5.9 mAb, the 5.10 mAb, or normalmouse IgG, all also coated (at a concentration of 10 μg/ml) onto thewell-bottoms of the 96-well tissue culture plates. ³H-TdR incorporationmeasured after a 48-hour culture period was used to determine T cellproliferation. (FIG. 9). Both anti-human 4-1BB stimulated significant Tcell proliferation, thereby demonstrating the T cell costimulatorypotential of these antibodies.

Example 9 MAb Enhances the Therapeutic Effect of RecombinantGM-CSF-Expressing Irradiated Tumor Cell Vaccine

Experiments were performed in order to determine whether an agonisticmAb specific for murine 41-BB (mAb 2A) enhances the anti-metastaticimmune response stimulated by irradiated tumor cells expressing arecombinant cytokine. Four groups of five B6 mice were injected i.v.with 5×10⁵ B16-F10 melanoma cells in order to generate disseminatedmetastases. On days 3, 7, and 11 the mice were immunized s.c. with 4×10⁶irradiated B16-F10 cells stably transfected with and expressing cDNAencoding murine GM-CSF (B16-GM-CSF) and on days 4, 7, 10, 12, and 15 themice injected i.p. with 100 μg of either rat IgG or mAb 2A as indicatedbelow. The B16-GM-CSF cells were generated essentially as described inDranoff et al. (1993) Proc. Natl. Acad. Sci. USA 90:3539-3543.

Group 1: rat IgG only

Group 2: mAb 2A only

Group 3: B16-GM-CSF and rat IgG

Group 4: B16-GM-CSF. and mAb 2A

The mice were monitored for survival (FIG. 10). Mice immunized withB-16-GM-CSF and mAb 2A showed enhanced survival over mice immunized withB16-GM-CSF and rat IgG.

Example 10 Induction of Anergy in CD8⁺ OT-1 T cells FollowingIntravenous Administration of OVA Peptide

Intravenous (i.v.) delivery of antigen is an established approach toinduce anergy in CD4⁺ T cells [Valujskikh et al. (2001) Transplantation72:685-693; Thorstenson et al. (2001) J. Immunol. 167:188-195; andBercovici et al. (1999) Eur. J. Immunol. 29:345-354]. Following i.v.injection of antigen, antigen-specific T cells exposed to a high dose ofsoluble antigen become unresponsive to the antigen; this is demonstratedby the inability of the T cells to proliferate and secrete IL-2 uponexposure to the antigen in vitro in the presence of APC [Jacobs et. al.(1994) Immunology 82:294-300]. This approach was adapted by theinventors to examine the induction of anergy in CD8⁺ T cells.

The intravenous administration of a H-2K^(b)-restricted peptide epitopeof ovalbumin (the “OVA peptide”) [Deeths et al. (1999) J. Immunol.163:102-110] to B6 mice to which OT-1 T cells had previously beenadoptively transferred, led to the activation and clonal expansion ofthe OT-1 T cells. Two days after peptide administration, pooled lymphnode and spleen cells from mice that had received a control peptide orthe OVA peptide were stained with both anti-CD8 antibody and tetramericH-2K^(b)-OVA (OVA tetramer). Whereas only 2.5% of the CD8⁺ T cells wereOVA tetramer-positive (i.e., were OT-1 T cells) in the mice treated withcontrol peptide, the proportion of CD8⁺ T cells that were OT-1 T cellsin the OVA-treated mice was 6.8% (FIG. 11). OT-1 cell blastogenesis wasalso observed in the OVA-treated mice, as demonstrated by the increasein forward scatter of the cells from these mice by FACS analysis.Furthermore, more than 50% of the OT-1 cells in the OVA-treated miceexpressed the T-cell activation markers, CD69 and CD25 (FIG. 11).

To determine the responsiveness of OT-1 T cells after i.v. exposure toOVA peptide, B6 mice treated as described above were rechallenged i.v.10 days later with either a control peptide or the OVA peptide. Forcomparison, naïve mice (i.e., mice that had not received the initialinjection of OVA peptide) were also injected with either the controlpeptide or OVA peptide. Two days following peptide administration,spleens and lymph nodes were harvested from the mice and the totalnumbers of OT-1 cells in pooled spleen and lymph node samples weredetermined by OVA tetramer staining. A greater than 4-fold expansion wasobserved in the naïve mice treated with OVA. In contrast, no significantexpansion of OT-1 cells resulted from rechallenge with OVA in those micethat had received OVA ten days previously (FIG. 12A), thus suggestingthe induction of OT-1 T cell anergy by the intial exposure to OVA.Unlike naïve OT-1 T cells, in which the expression of CD69 and CD25could be induced following antigenic challenge, the majority of anergicOT-1 T cells failed to express significant levels of CD25 followingantigenic challenge, although significant expression of CD69 wasobserved (FIG. 12B).

In order to determine whether or not OT-1 cells retained their effectorfunction following the induction of anergy, splenocytes from B6 miceinjected with OT1 T cells and a single dose of OVA as described abovewere restimulated with OVA peptide in vitro. Although the OT-1 cellspre-exposed to OVA were incapable of proliferating in vivo, theysecreted IFN-γ upon restimulation with OVA in vitro at a level similarto naïve OT-1 T cells (FIG. 12C). These results indicate that anergicOT-1 T cells retained the ability to secrete IFN-γ upon restimulation.

Example 11 CD137 Signaling Prevents the Induction of OT-1 T Cell Anergy

Following adoptive transfer of OT-1 cells into naïve B6 mice and theadministration of OVA peptide, mice were injected i.p. with either aCD137 mAb (clone 2A) or control rat IgG. Mice were sacrificed at varioustime points up to 21 days following injection of the OVA peptide and thetotal number of OT-1 cells present in the spleens and lymph nodes ineach group of mice was determined by OVA tetramer staining. As shown inFIG. 13A, treatment with anti-CD137 mAb following OVA administration ledto an approximately ten-fold increase in the number OT-1 cells comparedto those mice that received the control rat IgG. This robust T cellresponse following CD137 mAb injection led to significant splenomegalyin the CD137 mAb treated mice (data not shown). In contrast, the T cellresponse in the mice given the control rat IgG peaked 2 days followingOVA administration and rapidly declined, reaching baseline by day 21.OT-1 cells in the anti-CD137 mAb treated mice persisted for at least 21days following antigenic stimulation. These data demonstrate the abilityof CD137 stimulation to promote the expansion of OT-1 T cells in vivo.

To determine the effect of CD 137 signaling on induction of T cellanergy, mice to which OT-1 T cells had been adoptively transferred andthat were injected with either PBS, OVA peptide and anti-CD137 mAb, orOVA peptide and control rat IgG were sacrificed ten days followingpeptide (or PBS) administration. The number of OT-1 cells present in thepooled splenocytes from each group of mice was determined by OVAtetramer staining. The splenocytes were subsequently restimulated invitro with an optimal concentration of OVA peptide. OT-1 proliferationand IL-2 secretion were measured 72 and 48 hours later, respectively.The proliferation of OT-1 T cells, as measured by [³H]-thymidineincorporation, was determined on a per cell basis. Unlike the naïve OT1T cells from mice that had received PBS, OT-1 cells from mice that weregiven the OVA peptide and control rat IgG failed to proliferatefollowing in vitro restimulation (FIG. 13B). Furthermore, FACS analysisdemonstrated that virtually 100% of the OT-1 T cells from the micepreviously exposed to OVA peptide expressed the late activation markerVLA-4, in contrast to the OT-1 cells in the control peptide-treated mice(FIG. 13C). In sharp contrast, proliferation was observed in OT-1 Tcells from those mice that received anti-CD137 mAb and OVA peptide. IL-2production was not observed following restimulation of the anergic OT-1cells from OVA peptide and anti-CD137 mAb treated mice, a findingconsistent with their lack of proliferation (FIG. 13B) whereas OT-1cells from the anti-CD137 treated mice secreted IL-2 to an extentcomparable with naïve OT-1 (FIG. 3B); this finding provided furtherevidence that CD137 signaling prevented the induction of T cell anergy.

Example 12 CD137 Signaling Reverses OT-1 T Cell Anergy

Experiments were performed to determine whether CD137 signaling couldreverse anergy in an already anergic T cell. To address this question,naïve B6 to which OT-1 T cells had been adoptively tranferred mice wereinjected i.v. with OVA peptide as described above in order to induceanergy in the OT-1 T cells. Ten days later, the mice were injected i.p.with anti-CD137 mAb together with OVA peptide. Mice were sacrificed 2, 5and 10 days following treatment and the total number of OT-1 T cells wasdetermined by OVA tetramer staining. As shown in FIG. 14A, a markedexpansion of the OT-1 cells was observed in OVA-exposed mice that weretreated with anti-CD137 mAb and OVA peptide. The anergic OT-1 cells,however, failed to expand following a subsequent challenge with eitherthe OVA peptide or control peptide (FIG. 14A). These findings indicatethat CD137 signaling, in addition to preventing the induction of T cellanergy, breaks anergy in OT-1 T cells that had been previouslyanergized.

While the above results indicate that anti-CD137 mAb together with OVApeptide can reverse anergy in OT-1 T cells, it is unclear whethersignaling via the T cell receptor is absolutely necessary for anti-CD137mAb to break anergy. To test this, the OVA peptide OT-1 T cell anergicmice were prepared as described above. Ten days after the injection ofOVA peptide, the mice were given various combinations of control peptideor OVA peptide and control rat IgG or anti-CD137 mAb. Three days later,the mice were sacrificed and the total number of OT-1 cells determined.OT-1 cell expansion was only observed in the group of mice that receivedboth OVA peptide and anti-CD137 mAb (FIG. 14B). Importantly, no OT-1 Tcell proliferation was observed in the group of mice that received thecontrol peptide and anti-CD137 mAb. As was shown above (FIG. 12B),anergic OT-1 cells failed to express CD25 following encounter withantigen. However, CD137 signaling restored, at least in part, theability of these cells to express CD25 (FIG. 14C). These data show thatreversal of anergy in CD8⁺ T cells by CD137 mAb requires TCR engagement.

Experiments were then carried out to test whether cytolytic activity ofOT-1 T cells is retained by CD137 mAb exposure. OT-1 cells were sortedby FACS following staining with the OVA tetramer from the OVA-tolerizedmice 5 days after challenge with OVA peptide and CD137 mAb and were usedas effector cells in a standard 4-hour ⁵¹Cr-release assay forcytotoxicity against OVA-pulsed EL4 and control EL4 cells (FIG. 14D;“Anergic (OVA+2A)”). As controls, OT-1 cells were identically sortedfrom mice that, instead of initially being injected with the OVApeptide, were injected with the control peptide and then 10 days laterwere challenged with the OVA peptide and either rat IgG (“OVA+rat IgG”)or anti-CD137 mAb (“OVA+2A”); the cell sorting cytotoxicity testing, asfor the experimental group, were performed 5 days after the peptidechallenge and mAb/control IgG treatment. As shown in FIG. 14D,approximately 20% specific lysis was observed at an E:T ratio of 10 to 1in those mice that had received the control IgG. However, a more thantwo-fold increase in cytotoxicity.was observed in OT-1 cells isolatedfrom those mice that had received anti-CD137 mAb. Importantly, a similarlevel of cytotoxicity was observed in OT-1 cells isolated from both thenaïve and the tolerized mice, provided they received anti-CD137 mAb,indicating that anti-CD137 mAb not only restores proliferative capacity,but also cytotoxicity, in previously anergized OT-1 cells.

Example 13 CD137 Signaling in the Prevention and Reversal of P1APeptide-induced Tolerance in Tumor-specific T cells

A previously established tumor model was used to examine the effect ofCD137 signaling in the prevention and reversal of T cell tolerance.P815R is a clonal variant of highly tumorigenic P815 mastocytoma, whichunlike the parent P815 tumor, regresses spontaneously after inoculationinto syngeneic DBA/2 mice [Nieland et al. (1999) J. Cell Biochem.73:145-1521]. Administration of a peptide epitope of P1A, a non-mutatedself tumor antigen [Van den Eynde et al. (1991) J. Exp. Med.173:1373-1384], in incomplete Freund's adjuvant (IFA) inducesunresponsiveness of CD8⁺ T cells and promotes the progressive growth ofthe P815R tumor [Nieland et at. (1999) J. Cell Biochem. 73:145-152].Subcutaneous injection of P815R cells into DBA/2 mice led to thetransient development of subcutaneous tumors.

At day 40 after tumor inoculation, 80% of the mice that were treatedwith IFA alone were tumor-free (FIG. 15A, left panel). In contrast, only10% of mice were tumor free following the administration of P1A peptidewith control rat IgG ten days prior to tumor challenge (FIG. 15A, middlepanel), suggesting that the injection of P1A peptide induced tolerancein P1A-specific CD8⁺ CTL. Treatment with anti-CD137 mAb together withP1A peptide prevented tumor development altogether in 50% of mice (FIG.15A, right panel); however, tumor regression was observed in 40% of themice that did develop tumors, suggesting that treatment with anti-CD 137mAb prevents the induction of P1A-induced T cell anergy.

To test for an effect of anti-CD137 mAb on the regression of establishedP815R tumor caused by P1A peptide-induced anergy, mice were firsttolerized with P1A peptide in IFA for 10 days and were subsequentlychallenged with P815R cells. Three days following tumor challenge, micewere given control rat IgG or anti-CD 137 mAb. In mice treated with thecontrol rat IgG 90% of the mice developed progressively growing tumors(FIG. 15B, left panel). In contrast, anti-CD137 mAb treatment led totumor regression in 100% of mice (FIG. 15B, right panel). These findingsindicate that CD137 signaling can prevent and break T cell anergy afterthe administration of a tolerogenic P1A peptide, leading to theregression of P815R tumors.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method of generating an enhanced immune response in a subject, the method comprising administering to the subject: (a) an immunogenic stimulus, wherein the immunogenic stimulus comprises a polypeptide and does not comprise a tumor cell; and (b) an agonistic antibody that binds to 4-1BB.
 2. The method of claim 1, wherein the immunogenic stimulus is (a) a tumor-associated antigen (TAA) or (b) a functional fragment of a TAA.
 3. The method of claim 2, wherein the TAA is a molecule produced by a cancer cell selected from the group consisting of a leukemia, a lymphoma, a neurological cancer, a melanoma, a breast cancer, a lung cancer, a head and neck cancer, a gastrointestinal cancer, a liver cancer, a pancreatic cancer, a genitourinary cancer, a prostate cancer, a renal cell cancer, a bone cancer, and a vascular cancer cell.
 4. The method of claim 1, wherein the immunogenic stimulus is a molecule produced by an infectious microorganism.
 5. The method of claim 4, wherein the infectious microorganism is a virus.
 6. The method of claim 5, wherein the virus is a retrovirus.
 7. The method of claim 4, wherein the infectious microorganism is selected from the group consisting of a bacterium, a fungus, and a protozoan parasite.
 8. The method of claim 1, wherein the subject is a human.
 9. The method of claim 1, wherein the immune response is a response of a T cell.
 10. The method of claim 9, wherein the T cell is a CD8+ T cell.
 11. The method of claim 9, wherein the T cell is CD4+ T cell.
 12. The method of claim 1, wherein the immunogenic stimulus is a TAA, a peptide-epitope of a TAA, or a heat shock protein bound to peptide-epitope of a protein expressed by a tumor cell.
 13. An in vitro method of activating a T cell, the method comprising: (a) providing a cell sample comprising a T cell; and (b) culturing the cell sample with an immunogenic stimulus and an agonistic 4-1BB-binding agent.
 14. A method of preventing induction of energy or of reversing anergy in a T cell, the method comprising contacting the T cell with: (a) an immunogenic stimulus, wherein the immunogenic stimulus comprises a polypeptide and does not comprise a tumor cell; and (b) an agonistic antibody that binds to 4-1BB.
 15. The method of claim 14, wherein the contacting is in vitro.
 16. The method of claim 14, wherein the T cell is in a mammal.
 17. The method of claim 16, wherein the mammal is a human.
 18. The method of claim 16, wherein the contacting comprises administering to the mammal: (a) the immunogenic stimulus and the agonistic antibody; or (b) the immunogenic stimulus and a nucleic acid encoding the agonistic 4-1BB antibody.
 19. The method of claim 18, wherein the method comprises administering a cell transfected or transduced with a nucleic acid encoding the antibody to the mammal, wherein the cell is a cell, or a progeny of a cell, that prior to the transfection or the transduction, was obtained from the mammal.
 20. The method of claim 14, wherein the immunogenic stimulus is (a) a tumor-associated antigen (TAA) or (b) a functional fragment of a TAA.
 21. The method of claim 20, wherein the TAA is a molecule produced by a cancer cell selected from the group consisting of a leukemia, a lymphoma, a neurological cancer, a melanoma, a breast cancer, a lung cancer, a head and neck cancer, a gastrointestinal cancer, a liver cancer, a pancreatic cancer, a genitourinary cancer, a prostate cancer, a renal cell cancer, a bone cancer, and a vascular cancer cell.
 22. The method of claim 14, wherein the immunogenic stimulus is a molecule produced by an infectious microorganism.
 23. The method of claim 22, wherein the infectious microorganism is a virus.
 24. The method of claim 23, wherein the virus is a retrovirus.
 25. The method of claim 22, wherein the infectious microorganism is selected from the group consisting of a bacterium, a fungus, and a protozoan parasite.
 26. The method of claim 14, wherein the T cell is a CD8+ T cell.
 27. The method of claim 14, wherein the T cell is CD4+ T cell.
 28. The method of claim 14, wherein the immunogenic stimulus is a TAA, a peptide-epitope of a TAA, or a heat shock protein bound to peptide-epitope of a protein expressed by a tumor cell. 